这项工作是通过维多利亚州政府业务基础设施支助和澳大利亚政府NHMRC IRIISS实现的，并得到国家卫生和医学研究理事会（ALW #1120300）和澳大利亚研究理事会（KJH #FT190100265）的资助。作者希望感谢莫纳什动物研究平台、莫纳什组织学平台和莫纳什微成像设施的技术支持。

卵巢是从雌性C57BL6J小鼠身上采集的。所有动物程序和实验均按照《NHMRC澳大利亚动物护理和使用业务守则》进行，并经莫纳什动物研究平台动物伦理委员会批准。
注：本报告使用了一种化疗剂，表明其耗尽了原始卵泡卵母细胞，根据确定使用立体学11和直接计数12，13来比较同一动物的两种计数方法。雌性8周大（年轻成人）小鼠在单次肾内注射75毫克/千克/体重的环磷酰胺或盐水车控制（n=5/组）之前称体重。这种剂量已被证明会导致原始卵泡约50%的耗竭，但没有报告导致发病率或死亡率的小鼠14。卵巢在治疗后48小时收获。每只动物的一个卵巢固定在10%（v/v）中性缓冲形式素溶液中24小时，另一个固定在Boin的溶液中24小时。然后，组织被嵌入甘油甲基酸酯树脂中，并以20微米连续分割，或在石蜡中连续分割，在5微米处连续分割。所有组织都沾染了周期性酸希夫和血氧林。
1. 组学准备：固定、处理、嵌入和分割小鼠卵巢
<ol><li>通过修剪卵巢和周围所有脂肪来解剖小鼠卵巢，而不会损坏或切割卵巢本身。如有必要，使用解剖显微镜和细刀片进行此步骤（图 1A）。</li>
	<li>立即修复组织，放入一个小标签管，其中含有Boin的固定（立体学），或形式固定（直接计数）24小时（图1B），然后将组织转移到70%乙醇。 注：卵泡形态在布因的固定卵巢组织内保存得最好，嵌入到甘油甲酸酯树脂中（图2）。</li>
	<li>处理整个固定卵巢，然后嵌入糖甲基丙烯酸酯树脂用于立体学（补充文件1），或石蜡直接计数使用标准组织学协议。 注意：树脂是有毒的，所以确保所有组织处理步骤都用烟熏罩进行，手套随时佩戴。</li>
	<li>使用装有玻璃刀（图1D）的专用树脂甲基酸酯微原子（图1C）彻底切割厚糖甲基丙烯酸酯树脂部分（如20微米）。定期将各部分（例如，每3 节）收集到玻璃显微镜幻灯片上，用于立体学。</li>
	<li>使用标准微原子切割薄石蜡部分（例如，4-6 μm）。定期将组织部分（例如，每9 节 ）收集到玻璃显微镜幻灯片上，以进行直接的毛囊计数。</li>
	<li>用周期性酸希夫和血氧林（补充文件2）染色幻灯片。</li>
	<li>覆盖唇与标准DPX石蜡部分，或厚DPX甘油甲酸酯树脂部分（图1E）。 注意： 甘油甲酸酯树脂 DPX 是危险的， 所以执行这一步骤在烟雾罩. 注：甘油甲酸酯树脂DPX是非常粘稠的。玻璃盖片必须用铲子将其按下，以确保 DPX 均匀且分散，并且盖片下不存在气泡（图 1F）。</li>
</ol>2. 使用光学分馏器对原始卵泡数进行类固理估计
<ol><li>打开计算机、多控制单元、相机和立体学设置中的光源，将显微镜目标设置为低倍率（例如，10 倍）。</li>
	<li>打开立体声软件。</li>
	<li>将第一张幻灯片安全地放在显微镜舞台上。</li>
	<li>通过检查相机设置面板中的自动曝光（补充图 1A）来调整光线照射。或者，手动调整光线照射。</li>
	<li>使用操纵杆定位第一个组织样本，并将样品对焦。</li>
	<li>通过单击"更多设置"（位于相机设置面板的右下角）来调整白平衡，然后单击"白平衡"并单击"自动"（辅助图 1B）。或者，单击"更多设置"按钮（或"更多设置中的选择区域"）旁边的"白平衡"按钮，通过选择该部分上的白色区域手动设置白平衡。</li>
	<li>转到 探头 下拉菜单，然后单击 光学分数器工作流。然后单击 "开始新项目" 并单击 "确定"。
	<ol>
<li>如果已保存现有采样配置，请在 采样参数 下单击 "是 "| ... 并选择所需的采样配置。</li>
		<li>如果没有，单击 "否" 并手动输入串行部分信息（补充图 1C），并在步骤 2.13 中定义探头配置。</li>
	</ol></li>
	<li>单击 "下一步"，将显微镜设置为 "低放大"， 并从下拉菜单中选择 10 倍 的放大倍数。</li>
	<li>单击 "下一步"，然后跟踪整个卵巢部分 - 从左侧单击该部分开始，到末尾，右键单击并选择 "关闭轮廓"。</li>
	<li>单击 "下一步"，将显微镜设置为 高放大， 并从下拉菜单中选择 100 倍的油放大倍率 。</li>
	<li>将一滴油放在滑梯上的组织部分，并将显微镜目标移动至 100 倍放大。</li>
	<li>调整光线照射（如步骤 2.4），然后单击 "下一步"。</li>
	<li>设置 采样参数 以定义探头配置。在这里，计数框架设置为 47.5 μm x 47.5 μm （2，256.25 μm2），步长设置为 100μm x 100 μm （10，000 μm2）（补充图 2）。建立采样参数后，保存模板，并在后续分析过程中以步骤 2.7 重新打开。</li>
	<li>点击 开始计数 （补充图1D）。聚焦到示例顶部，单击 "确定" 并开始计数。</li>
	<li>使用聚焦旋钮穿过 10 μm 采样深度，并计算其卵母细胞核进入焦点的任何原始卵泡。单击 "下一步 "移动到下一个区域。<ol>
<li>如果卵母细胞被鳞状（扁平）颗粒细胞包围，则将卵泡分类为原始卵泡，但没有阴囊颗粒细胞（图2A）。 注：原始卵泡不同于中叶/过渡卵泡，由胆囊和鳞状粒状细胞（图2B）和原卵泡组成，主要由阴囊状毛囊细胞（图2C）包围。这些卵泡类应单独量化。</li>
		<li>仅计算卵母细胞核可见的卵泡。卵母细胞核必须出现在计数帧内或触摸计数帧的绿色内含线（补充图1E，F）。</li>
		<li>如果卵母细胞核落在计数帧（补充图1G）之外或触及计数帧的红色排除线，则不要计算此毛囊。</li>
		<li>在评估原始卵泡消耗以响应外源性化学物质（如化疗）时，确保所有计算的卵泡都健康，从而具有正常的形态（图 2）。单独计算任何异常或节觉卵泡。通常，卵泡死亡是由化疗等侮辱引起的，为了区分健康和不健康的卵泡，应单独获得对隐性卵泡的定量，因为只有健康的卵泡才包括卵巢储备15。</li>
	</ol></li>
	<li>在该部分完成计数后，进行以下部分之一：<ol>
<li>单击 "添加新节"，然后返回到步骤 2.3 以设置下一节进行计数。</li>
		<li>单击 "我已完成计数 以结束会话"。将目标返回到 10 倍，退出立体声软件，关闭光源、摄像头、多控制单元和计算机。</li>
	</ol></li>
	<li>从从整个卵巢取样的每个组织部分获取总和原始卵泡编号（Q），然后使用电子表格，使用下面的方程从每个复制动物 （N）4中获取最终值。 <img alt="Equation 1" src="/files/ftp_upload/61782/61782equ01.jpg">哪里： N = 卵巢内卵泡的总估计数量。 Q= 原始卵泡计数。 f1= 采样间隔。因此，每3节 进行采样 <img alt="Equation 2" src="/files/ftp_upload/61782/61782equ02.jpg"> 。 f2 = 计数帧（示例区域）和步进器之间的关系，按 <img alt="Equation 3" src="/files/ftp_upload/61782/61782equ03.jpg"> .由于样本面积为 2256 μm 2（47.5 μm x 47.5 μm），步进区域为 10000 μm2（100μm x 100 μm）。 <img alt="Equation 4" src="/files/ftp_upload/61782/61782equ04.jpg"> f3= 抽样卵巢部分的分数。由于20μm部分的10μm被采样， <img alt="Equation 5" src="/files/ftp_upload/61782/61782equ05.jpg"> . 因此， <img alt="Equation 6" src="/files/ftp_upload/61782/61782equ06.jpg"> . 注：本协议描述了如何使用广泛引用的立体学软件（材料表）应用这些固态分析原理：但是，还有其他的固态软件可用。卵巢卵泡的固态分析中应用的原则是相同的，无论用于设置参数的软件如何。当100个或更多物体计入成年小鼠卵巢4时，立体学是最准确的，因为这给出了一个误差系数，估计低于10%16。此处概述的采样参数已优化，以确保至少 100 个物体（即原始、过渡和原卵泡）可计入控制成人野生型 C57BL6J 卵巢。可以进行试点研究，包括小样本量，以确定最佳采样参数，如要分析的分区的间隔和数量，以及样本第17节内的光学组件数量。</li>
</ol>3. 通过直接卵巢卵泡计数估计原始卵泡数
<ol><li>将显微镜安全地滑动在标准光显微镜下，并进行直接计数以获取原始卵泡编号。<ol>
<li>如果卵母细胞清晰可见，并被鳞状（扁平）颗粒细胞包围，但无胆囊颗粒细胞（图2D），则将卵泡分类为原始卵泡。 注：原始卵泡不同于中叶/过渡卵泡，由胆囊和鳞状粒状细胞（图2E）和原卵泡组成，主要由阴囊颗粒细胞（图2F）包围。这些卵泡类应单独量化。</li>
		<li>确保所有计算的卵泡都健康，从而具有正常的形态（图2）。单独计算任何异常或节觉卵泡，因为只有健康的卵泡构成卵巢储备。</li>
	</ol>
</li>
	<li>或者，通过打开图像文件（s）来获取整个卵巢组织部分的多个或缝合的高功率（例如，20倍）光微镜来执行计数。这可以手动完成，也可以使用自动幻灯片扫描仪。</li>
	<li>在预定的间隔内从从整个卵巢取样的每个组织部分获取总和原始卵泡编号 （Q）。将此数字乘以采样分数的反向，以便使用以下方程获取每个复制动物 （N） 的最终值。 <img alt="Equation 7" src="/files/ftp_upload/61782/61782equ07.jpg">哪里： N = 卵巢内卵泡的总估计数量。 Q = 原始毛囊计数（每个单独类型的，单独计算）。 f1= 采样间隔。因此，每9节 进行采样 <img alt="Equation 8" src="/files/ftp_upload/61782/61782equ08.jpg"> 。 因此， <img alt="Equation 9" src="/files/ftp_upload/61782/61782equ09.jpg"> .</li>
</ol>

<ol>
	<li>Stringer, J. M., Winship, A., Liew, S. H., Hutt, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+capacity+of+oocytes+for+DNA+repair.">The capacity of oocytes for DNA repair.</a> Cellular and Molecular Life Sciences. 75 (15), 2777-2792 (2018).</li><li>Winship, A. L., Stringer, J. M., Liew, S. H., Hutt, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+DNA+repair+for+maintaining+oocyte+quality+in+response+to+anti-cancer+treatments,+environmental+toxins+and+maternal+ageing.">The importance of DNA repair for maintaining oocyte quality in response to anti-cancer treatments, environmental toxins and maternal ageing.</a> Human Reproduction Update. 24 (2), 119-134 (2018).</li><li>Broer, S. L., Broekmans, F. J., Laven, J. S., Fauser, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-Mullerian+hormone:+ovarian+reserve+testing+and+its+potential+clinical+implications.">Anti-Mullerian hormone: ovarian reserve testing and its potential clinical implications.</a> Human Reproduction Update. 20 (5), 688-701 (2014).</li><li>Myers, M., Britt, K. L., Wreford, N. G., Ebling, F. J., Kerr, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+quantifying+follicular+numbers+within+the+mouse+ovary.">Methods for quantifying follicular numbers within the mouse ovary.</a> Reproduction. 127 (5), 569-580 (2004).</li><li>Boyce, R. W., Dorph-Petersen, K. A., Lyck, L., Gundersen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design-based+stereology:+introduction+to+basic+concepts+and+practical+approaches+for+estimation+of+cell+number.">Design-based stereology: introduction to basic concepts and practical approaches for estimation of cell number.</a> Toxicologic Pathology. 38 (7), 1011-1025 (2010).</li><li>Ristic, N., 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=Maternal+dexamethasone+treatment+reduces+ovarian+follicle+number+in+neonatal+rat+offspring.">Maternal dexamethasone treatment reduces ovarian follicle number in neonatal rat offspring.</a> Journal of Microscopy. 232 (3), 549-557 (2008).</li><li>Soleimani Mehranjani, M., Mansoori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereological+study+on+the+effect+of+vitamin+C+in+preventing+the+adverse+effects+of+bisphenol+A+on+rat+ovary.">Stereological study on the effect of vitamin C in preventing the adverse effects of bisphenol A on rat ovary.</a> International Journal of Reproductive Biomedicine (Yazd). 14 (6), 403-410 (2016).</li><li>Brown, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bias+in+image+analysis+and+its+solution:+unbiased+stereology.">Bias in image analysis and its solution: unbiased stereology.</a> Journal of Toxicologic Pathology. 30 (3), 183-191 (2017).</li><li>Hasselholt, S., Lykkesfeldt, J., Overgaard Larsen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thick+methacrylate+sections+devoid+of+lost+caps+simplify+stereological+quantifications+based+on+the+optical+fractionator+design.">Thick methacrylate sections devoid of lost caps simplify stereological quantifications based on the optical fractionator design.</a> Anatomical Record (Hoboken). 298 (12), 2141-2150 (2015).</li><li>Tilly, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+follicle+counts--not+as+simple+as+1,+2,+3.">Ovarian follicle counts--not as simple as 1, 2, 3.</a> Reproductive Biology and Endocrinology. 1, 11 (2003).</li><li>Nguyen, Q. N., 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=Loss+of+PUMA+protects+the+ovarian+reserve+during+DNA-damaging+chemotherapy+and+preserves+fertility.">Loss of PUMA protects the ovarian reserve during DNA-damaging chemotherapy and preserves fertility.</a> Cell Death and Disease. 9 (6), 618 (2018).</li><li>Meirow, D., Assad, G., Dor, J., Rabinovici, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GnRH+antagonist+cetrorelix+reduces+cyclophosphamide-induced+ovarian+follicular+destruction+in+mice.">The GnRH antagonist cetrorelix reduces cyclophosphamide-induced ovarian follicular destruction in mice.</a> Human Reproduction. 19 (6), 1294-1299 (2004).</li><li>Winship, A. L., 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=The+PARP+inhibitor,+olaparib,+depletes+the+ovarian+reserve+in+mice:+implications+for+fertility+preservation.">The PARP inhibitor, olaparib, depletes the ovarian reserve in mice: implications for fertility preservation.</a> Human Reproduction. , (2020).</li><li>Meirow, D., Lewis, H., Nugent, D., Epstein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subclinical+depletion+of+primordial+follicular+reserve+in+mice+treated+with+cyclophosphamide:+clinical+importance+and+proposed+accurate+investigative+tool.">Subclinical depletion of primordial follicular reserve in mice treated with cyclophosphamide: clinical importance and proposed accurate investigative tool.</a> Human Reproduction. 14 (7), 1903-1907 (1999).</li><li>Nguyen, Q. N., 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=Cisplatin-+and+cyclophosphamide-induced+primordial+follicle+depletion+is+caused+by+direct+damage+to+oocytes.">Cisplatin- and cyclophosphamide-induced primordial follicle depletion is caused by direct damage to oocytes.</a> Molecular Human Reproduction. , (2019).</li><li>Gundersen, H. J., Jensen, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficiency+of+systematic+sampling+in+stereology+and+its+prediction.">The efficiency of systematic sampling in stereology and its prediction.</a> Journal of Microscopy. 147, 229-263 (1987).</li><li>Olesen, M. V., Needham, E. K., Pakkenberg, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Optical+Fractionator+Technique+to+Estimate+Cell+Numbers+in+a+Rat+Model+of+Electroconvulsive+Therapy.">The Optical Fractionator Technique to Estimate Cell Numbers in a Rat Model of Electroconvulsive Therapy.</a> Journal of Visualized Experiments. (125), e55737 (2017).</li><li>Findlay, J. K., Hutt, K. J., Hickey, M., Anderson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Is+the+Number+of+Primordial+Follicles+in+the+Ovarian+Reserve+Established.">How Is the Number of Primordial Follicles in the Ovarian Reserve Established.</a> Biology of Reproduction. 93 (5), 111 (2015).</li><li>Omari, 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=Mcl-1+is+a+key+regulator+of+the+ovarian+reserve.">Mcl-1 is a key regulator of the ovarian reserve.</a> Cell Death and Disease. 6, 1755 (2015).</li><li>Winship, A. L., Bakai, M., Sarma, U., Liew, S. H., Hutt, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dacarbazine+depletes+the+ovarian+reserve+in+mice+and+depletion+is+enhanced+with+age.">Dacarbazine depletes the ovarian reserve in mice and depletion is enhanced with age.</a> Scientific Reports. 8 (1), 6516 (2018).</li><li>Miller, P. B., Charleston, J. S., Battaglia, D. E., Klein, N. A., Soules, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+accurate,+simple+method+for+unbiased+determination+of+primordial+follicle+number+in+the+primate+ovary.">An accurate, simple method for unbiased determination of primordial follicle number in the primate ovary.</a> Biology of Reproduction. 56 (4), 909-915 (1997).</li><li>Miller, P. B., Charleston, J. S., Battaglia, D. E., Klein, N. A., Soules, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphometric+analysis+of+primordial+follicle+number+in+pigtailed+monkey+ovaries:+symmetry+and+relationship+with+age.">Morphometric analysis of primordial follicle number in pigtailed monkey ovaries: symmetry and relationship with age.</a> Biology of Reproduction. 61 (2), 553-556 (1999).</li><li>Charleston, J. 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=Estimating+human+ovarian+non-growing+follicle+number:+the+application+of+modern+stereology+techniques+to+an+old+problem.">Estimating human ovarian non-growing follicle number: the application of modern stereology techniques to an old problem.</a> Human Reproduction. 22 (8), 2103-2110 (2007).</li><li>Hansen, K. R., Craig, L. B., Zavy, M. T., Klein, N. A., Soules, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+primordial+and+nongrowing+follicle+counts+according+to+the+Stages+of+Reproductive+Aging+Workshop+(STRAW)+staging+system.">Ovarian primordial and nongrowing follicle counts according to the Stages of Reproductive Aging Workshop (STRAW) staging system.</a> Menopause. 19 (2), 164-171 (2012).</li><li>Hansen, K. R., 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+new+model+of+reproductive+aging:+the+decline+in+ovarian+non-growing+follicle+number+from+birth+to+menopause.">A new model of reproductive aging: the decline in ovarian non-growing follicle number from birth to menopause.</a> Human Reproduction. 23 (3), 699-708 (2008).</li><li>Sonigo, 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=High-throughput+ovarian+follicle+counting+by+an+innovative+deep+learning+approach.">High-throughput ovarian follicle counting by an innovative deep learning approach.</a> Scientific Reports. 8 (1), 13499 (2018).</li><li>McKey, J., Cameron, L. A., Lewis, D., Batchvarov, I. S., Capel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+iDISCO+and+CUBIC+tissue+clearing+and+lightsheet+microscopy+for+in+toto+analysis+of+the+adult+mouse+ovarydagger.">Combined iDISCO and CUBIC tissue clearing and lightsheet microscopy for in toto analysis of the adult mouse ovarydagger.</a> Biology of Reproduction. 102 (5), 1080-1089 (2020).</li><li>Kagami, K., Shinmyo, Y., Ono, M., Kawasaki, H., Fujiwara, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+evaluation+of+murine+ovarian+follicles+using+a+modified+CUBIC+tissue+clearing+method.">Three-dimensional evaluation of murine ovarian follicles using a modified CUBIC tissue clearing method.</a> Reproductive Biology and Endocrinology. 16 (1), 72 (2018).</li></ol>

作者没有什么可透露的。

本文为列举小鼠原始毛囊、立体学的金本位技术以及更常用的直接毛囊计数方法提供了分步协议。化疗用于比较和对比从同一动物的左右卵巢内这两种不同方法获得的结果。这两种方法都揭示了原始毛囊数量的高动物间变异性。使用立体学记录了卵巢储备的显著消耗，但直接计数未能检测到化疗与控制后原始卵泡数量显著减少。
值得注意的是，即使在近亲繁殖的老鼠，如C57BL...

储存在卵巢原始卵泡内的不成熟、被肌质逮捕的卵母细胞是雌性生殖系的储存单元，由个体的终身卵巢储备组成。原始毛囊数量随着1岁自然下降，或者，在接触外源性化学物质（包括空气、食物和水中的一些药物和环境毒剂）后，可过早耗尽。鉴于卵泡的原始数量有限，卵巢内卵泡的数量和质量在很大程度上决定了女性的生育能力和后代的健康。因此，准确量化妇女的原始卵泡数量对于评估外源性侮辱对卵巢储备的离目标影响至关重要。
在妇女中，对整个卵巢的分析通常是不可能的，因此卵巢储备的非侵入性代孕措施必须在临床环境中使用。抗Mϋllerian激素（AMH）是临床上应用最广泛的代孕生物标志物3。血清AMH水平通常测量在晚产妇年龄的妇女，或癌症治疗前后，如化疗。然而，AMH是由生长的卵泡而不是原始卵泡产生的，因此，血清水平不会告知绝对原始卵泡数量。
由于缺乏准确确定原位女性卵泡数量的方法，在啮齿动物等小型动物模型 中计算卵巢卵泡仍然是评估外源性侮辱对原始毛囊的影响程度以及生育能力的重要研究工具。然而不幸的是，在整个文献中，关于啮齿动物模型中原始卵泡数量的报道是高度可变的4。其主要原因是广泛报道的计数方法在技术上的差异。在文献中，主要描述了两种不同的技术来列举小鼠的原始卵泡。其中包括立体学，它采用了分馏器光学分解方法，以及直接卵泡计数。
立体学被广泛认为是金本位制，因为它使用系统均匀的随机抽样5，使其成为最准确的方法，量化原始卵泡数在整个小鼠，或大鼠卵巢4，6，7。立体学是公正的，因为它占了利益对象的三维结构。使用光学分解器/分孔器方法，使用三级采样方法，在已知小鼠总卵巢的一小部分内使用厚组织部分（例如 20 μm）对原始卵泡进行量化。首先，随机启动时选择采样间隔（例如，每3 节）（采样分数 1，f1）4。然后，从这一点到整个卵巢，以系统、均匀的方式对部分进行采样。然后，一个不偏不倚的计数框架叠加在卵巢部分，并逐步沿着一个定义的，随机计数网格（采样分数2，f2）8移动。最后，对该区段厚度的已知部分进行光学采样（例如，10μm），并计算该区域内的毛囊（采样分数 3，f3）4。原始毛囊数乘以这些采样分数的反向，以获得最终值。这种方法需要专家培训和设备，包括由固态软件驱动的具有机动阶段的显微镜。组织应保存在专门的Boin的固定剂中，并嵌入甘油甲酸酯树脂中，以便使用玻璃刀切割厚组织部分。这种方法旨在解释组织收缩和变形，以最好地保持卵巢和卵泡9的三维形态结构。
直接卵泡计数是计算卵泡10的最常用的方法。可以使用更常见的固定剂（即形式素），然后是石蜡嵌入和详尽的连续剖面，使用厚度在 4-6 μm 之间的标准微原子。卵泡在定义的间隔内系统地计入整个组织部分，然后乘以采样间隔的反向，以获得毛囊总估计值。这种方法快，简单，可以使用存档组织执行，并使用标准组织学技术准备。它只需要一台具有标准成像能力的光显微镜。然而，尽管有这些优点，直接卵泡计数缺乏立体学的准确性和严格的计数参数，使其更容易受到调查员的偏差。此外，组织在加工过程中可能会发生收缩和变形，破坏卵巢的完整性和形态，从而使卵泡分类和定量变得困难。
本文的目的是描述两种常用的方法，定量评估小鼠卵巢的原始卵泡数量：立体学和直接卵泡计数。我们将为这两种方法提供详细的协议，并突出它们的一些优点和短处，以提高我们领域的可重复性，并使研究人员能够做出最合适的计算方法的知情决定，以供其研究使用。

<table>
	<tbody>
		<tr>
			<td>1-Butanol (HPLC)</td>
			<td>Fisher Chemical</td>
			<td>#A383-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Acid alcohol</td>
			<td>Amber Scientific</td>
			<td>#ACDL</td>
			<td></td>
		</tr>
		<tr>
			<td>Bouin&#8217;s fixative</td>
			<td>Sigma-Aldrich</td>
			<td>#HT10132</td>
			<td>Picric acid 0.9% (w/v), formaldehyde 9% (v/v), acetic acid 5% (w/v)</td>
		</tr>
		<tr>
			<td>Cyclophosphamide</td>
			<td>Sigma-Aldrich</td>
			<td>#C0768-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dibutylphthalate Polystyrene Xylene (DPX)</td>
			<td>Sigma-Aldrich</td>
			<td>#06522</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Amber Scientific</td>
			<td>#ETH</td>
			<td>Ethanol 100%</td>
		</tr>
		<tr>
			<td>Micro Feather opthalmic scalpel with aluminium handle</td>
			<td>Designs for Vision</td>
			<td>#FEA-745-SR</td>
			<td>Feather blade for dissections (seen in Figure 1)</td>
		</tr>
		<tr>
			<td>Formalin fixative</td>
			<td>Australian Biostain</td>
			<td>#ANBFC</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslip</td>
			<td>Thermo Scientific</td>
			<td>#MENCS22501GP</td>
			<td>22 mm x 50 mm</td>
		</tr>
		<tr>
			<td>Glycomethacrylate resin RM2165 microtome</td>
			<td>Leica Microsystems</td>
			<td>#RM2165</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycolmethacrylate DPX</td>
			<td>*made in house</td>
			<td></td>
			<td>*Mix 1.5 L Xylene; 800 g polystyrene pellets; 100mL Dibutyl phthalate for 3 weeks</td>
		</tr>
		<tr>
			<td>Histolene</td>
			<td>Trajan</td>
			<td>#11031</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayer&#8217;s haematoxylin</td>
			<td>Amber Scientific</td>
			<td>#MH</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus BX50 microscope</td>
			<td>Olympus</td>
			<td>#BX50</td>
			<td>Brightfield microscope fitted with 10x dry &#38; 100x oil immersion objective (numerical aperture 1.3)</td>
		</tr>
		<tr>
			<td>Olympus immersion oil type-F</td>
			<td>Olympus</td>
			<td>#IMMOIL-F30CC</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus TH4-200 light source</td>
			<td>Olympus</td>
			<td>#TH4-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin wax</td>
			<td>Sigma-Aldrich</td>
			<td>#03987</td>
			<td></td>
		</tr>
		<tr>
			<td>Periodic acid</td>
			<td>Trajan</td>
			<td>#PERI1%</td>
			<td>Periodic acid 1%</td>
		</tr>
		<tr>
			<td>Rotary Microtome CUT 4060</td>
			<td>MicroTec</td>
			<td>#4060R/F</td>
			<td>Used to cut paraffin sections</td>
		</tr>
		<tr>
			<td>Schiff&#8217;s reagent</td>
			<td>Trajan</td>
			<td>#SCHF</td>
			<td></td>
		</tr>
		<tr>
			<td>Scott&#39;s tap water</td>
			<td>Amber Scientific</td>
			<td>#SCOT</td>
			<td>Potassium carbonate, magnesium sulphate, water</td>
		</tr>
		<tr>
			<td>StereoInvestigator Stereological System</td>
			<td>MBF Bioscience</td>
			<td></td>
			<td>Includes StereoInvestigator software, multi-control unit, automatic stage and joystick</td>
		</tr>
		<tr>
			<td>Superfrost microscope slides</td>
			<td>Thermo Scientific</td>
			<td>#MENSF41296SP</td>
			<td>1 mm, 72 pcs</td>
		</tr>
		<tr>
			<td>Technovit 7100 Plastic embedding system</td>
			<td>Emgrid Australia</td>
			<td>#64709003</td>
			<td>500 mL/5 x 1 g/40 mL</td>
		</tr>
		<tr>
			<td>Technovit 3040 yellow</td>
			<td>Emgrid Australia</td>
			<td>#64708805</td>
			<td>100 g/80 mL</td>
		</tr>
	</tbody>
</table>

accurate follicle enumeration in adult mouse ovaries

性繁殖的雌性哺乳动物生来就有一生供应的卵母细胞。不成熟、静默的卵母细胞存在于原始卵泡中，这是雌性生殖系的储存单元。它们不可再生，因此它们出生时的数量和随后的丧失率在很大程度上决定了女性的生育寿命。准确量化妇女和动物的原始卵泡数量对于确定药物和毒剂对卵巢保护区的影响至关重要。还需要评估现有和新兴生育保护技术的必要性和成功性。目前，没有方法可以准确测量构成妇女卵巢储备的原始卵泡数量。此外，从大型动物或濒危物种获得卵巢组织进行实验往往不可行。因此，小鼠已成为此类研究的基本模型，评估整个小鼠卵巢原始卵泡数量的能力是一个关键工具。然而，文献中关于小鼠卵巢绝对卵泡数的报告变化很大，因此很难比较和/或复制数据。这是由于许多因素，包括应变，年龄，治疗组，以及技术差异的计数方法使用。在本文中，我们提供分步教学指南，用于准备组发部分和使用两种不同的方法计算小鼠卵巢中的原始卵泡：[1] 立体学，它依赖于分馏器/光学分解技术：和[2]直接计数技术。将突出每种方法的一些关键优点和缺点，以便在该领域提高可重复性，使研究人员能够选择最适合其研究的方法。

使用了一种特征良好的卵泡消耗模型，即对年轻的成年雌性小鼠进行单剂量的环磷酰胺化疗，或盐水车辆控制（n=5/组），并在48小时后从每种动物身上采集两个卵巢。每只动物的卵巢是按照第1步为两种方法中描述的那样准备的：立体学或直接计数。每个动物的左右卵巢被随机分配给每组。这些数据表明，使用立体学时，在化疗后可以检测到小鼠原始卵泡的显著消耗（387±11个卵泡），而对照（1043?...

Watch this Scientific Journal Video about Accurate Follicle Enumeration in Adult Mouse Ovaries at JoVE.com

Accurate Follicle Enumeration in Adult Mouse Ovaries

在这里，我们描述，比较和对比两种不同的技术，以准确计算固定小鼠卵巢组织的卵泡计数。

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 ...

accurate-follicle-enumeration-in-adult-mouse-ovaries

Research

JoVE Journal

Developmental Biology

8.0K 次观看.  Monash University. 在这里，我们描述，比较和对比两种不同的技术，以准确计算固定小鼠卵巢组织的卵泡计数。

视频: Accurate Follicle Enumeration in Adult Mouse Ovaries

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

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

从背小鼠皮肤分离毛囊干细胞和表皮角质形成细胞

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

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

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

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

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

活体果蝇卵巢组织中卵泡干细胞谱系的细胞内 pH 值的成像方法

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

Dissection and Staining of Drosophila Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Dissection and Staining of Drosophila Pupal Ovaries

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

果蝇蛹卵巢的解剖与染色

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

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, ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

成人小鼠成纤维细胞对红细胞生成子细胞的直接线重编程

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and behave by observing the surface of a tissue. This method provides a means to visualize the complex 3-dimensional vascular architecture of the lung.
To accomplish this, a catheter is inserted into the pulmonary artery and the vasculature is simultaneously flushed of blood and chemically dilated to limit resistance. Lungs are then inflated through the trachea at a standard pressure and the polymer compound is infused into the vascular bed at a standard flow rate. Once the entire arterial network is filled and allowed to cure, the lung vasculature may be visualized directly or imaged on a micro-CT (&#181;CT) scanner.
When performed successfully, one can appreciate the pulmonary arterial network in mice ranging from early postnatal ages to adults. Additionally, while demonstrated in the pulmonary arterial bed, this method can be applied to any vascular bed with optimized catheter placement and endpoints.

The aim of this technique is ex vivo visualization of pulmonary arterial networks of early postnatal and adult mice through lung inflation and injection of a radio-opaque polymer-based compound via the pulmonary artery. Potential applications for casted tissues are also discussed.

用于微CT成像的成人和早期产后小鼠肺的血管铸造

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and ...

In Vitro Culture Strategy for Oocytes from Early Antral Follicle in Cattle

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that during the reproductive life span only about 1% of the oocytes in an ovary mature and ovulate, several techniques have been developed to increase the exploitation of the ovarian reserve to the growing population of non-ovulatory follicles. Such technologies have allowed interventions of fertility preservation, selection programs in livestock, and conservation of endangered species. However, the vast potential of the ovarian reserve is still largely unexploited. In cows, for instance, some attempts have been made to support in vitro culture of oocytes at specific developmental stages, but efficient and reliable protocols have not yet been developed. Here we describe a culture system that reproduce the physiological conditions of the corresponding follicular stage, defined to develop in vitro growing oocytes collected from bovine early antral follicles to the fully-grown stage, corresponding to the medium antral follicle in vivo. A combination of hormones and a phosphodiesterase 3 inhibitor was used to prevent untimely meiotic resumption and to guide oocyte&#39;s differentiation.

We describe the procedures for isolation of growing oocytes from ovarian follicles at early stages of development, as well as the setup of an in vitro culture system which can support the growth and differentiation up to the fully-grown stage. &#160;

牛早期蚁卵泡卵母细胞的体外培养策略

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that ...

Vitrification of In Vitro Matured Oocytes Collected from Adult and Prepubertal Ovaries in Sheep

In livestock, in vitro embryo production systems can be developed and sustained thanks to the large number of ovaries and oocytes that can be easily obtained from a slaughterhouse. Adult ovaries always bear several antral follicles, while in pre-pubertal donors the maximal numbers of oocytes are available at 4 weeks of age, when ovaries bear peak numbers of antral follicles. Thus, 4 weeks old lambs are considered good donors, even if the developmental competence of prepubertal oocytes is lower compared to their adult counterpart. 
Basic research and commercial applications would be boosted by the possibility of successfully cryopreserving vitrified oocytes obtained from both adult and prepubertal donors. The vitrification of oocyte collected from prepubertal donors would also allow shortening the generation interval and thus increasing the genetic gain in breeding programs. However, the loss of developmental potential after cryopreservation makes mammalian oocytes probably one of the most difficult cell types to cryopreserve. Among the available cryopreservation techniques, vitrification is widely applied to animal and human oocytes. Despite recent advancements in the technique, exposures to high concentrations of cryoprotective agents as well as chilling injury and osmotic stress still induce several structural and molecular alterations and reduce the developmental potential of mammalian oocytes. Here, we describe a protocol for the vitrification of sheep oocytes collected from juvenile and adult donors and matured in vitro prior to cryopreservation. The protocol includes all the procedures from oocyte in vitro maturation to vitrification, warming and post-warming incubation period. Oocytes vitrified at the MII stage can indeed be fertilized following warming, but they need extra time prior to fertilization to restore damage due to cryopreservation procedures and to increase their developmental potential. Thus, post-warming culture conditions and timing are crucial steps for the restoration of oocyte developmental potential, especially when oocyte are collected from juvenile donors.

The protocol aims at providing a standard method for the vitrification of adult and juvenile sheep oocytes. It includes all the steps from the preparation of the in vitro maturation media to the post-warming culture. Oocytes are vitrified at the MII stage using Cryotop to ensure the minimum essential volume.

从绵羊的成人和前卵巢收集的体外成熟卵母细胞的振动

In livestock, in vitro embryo production systems can be developed and sustained thanks to the large number of ovaries and oocytes that can be easily ...