人卵泡中的基因表达分析
Summary
在这里，我们描述了一个协议，概述了如何从冷冻解冻的皮质组织中分离人类卵巢卵泡以进行基因表达分析。
Abstract
卵巢是由不同细胞类型组成的异质器官。为了研究卵泡生成过程中发生的分子机制，可以在固定组织上进行蛋白质的定位和基因表达。然而，为了正确评估人类卵泡中的基因表达水平，必须分离这种复杂而微妙的结构。因此，Woodruff实验室先前描述的适应方案已被开发用于将卵泡（卵母细胞和颗粒细胞）与其周围环境分开。首先使用两种工具手动处理卵巢皮质组织以获得小碎片：组织切片机和组织切碎器。然后用0.2%胶原酶和0.02%DNA酶酶消化组织至少40分钟。该消化步骤在37°C和5%CO2 下进行，并伴随着每10分钟机械移液培养基。孵育后，在显微镜放大镜下使用校准的微毛细管移液管手动收集分离的卵泡。如果卵泡仍然存在于组织碎片中，则通过手动显微切割完成该过程。将卵泡收集在培养基中的冰上，并在磷酸盐缓冲盐溶液液滴中冲洗两次。必须仔细控制这种消化过程，以避免卵泡恶化。一旦卵泡的结构似乎受损或最多90分钟后，用含有10%胎牛血清的4°C封闭溶液停止反应。应收集至少20个分离的卵泡（大小小于75μm），以便在RNA提取后获得足够量的总RNA，以进行实时定量聚合酶链反应（RT-qPCR）。提取后，来自20个卵泡的总RNA的定量达到5ng / μL的平均值。然后将总RNA逆转录成cDNA，并使用RT-qPCR进一步分析感兴趣的基因。
Introduction
卵巢是由功能和结构单元组成的复杂器官，包括皮层内的滤泡和基质。卵泡发生，即卵泡活化、生长和成熟从原始静止状态到能够受精并支持早期胚胎发育的成熟卵泡的过程，在研究中被广泛研究1。揭示推动这一现象的机制可以改善妇女的生育护理2.对固定人体组织的分析可以评估卵巢功能单位内的蛋白质表达和基因定位3，4。然而，需要特定的技术将卵泡与周围的皮层分离，以准确评估卵巢卵泡内的基因表达水平。因此，在之前的一项研究中，开发了一种卵泡分离技术，允许直接从卵巢的功能单元分析基因表达5。已经开发了不同的方法，例如酶消化和/或机械分离，以及激光捕获显微切割，允许在一块组织内分离卵泡6，7，8，9。卵泡分离广泛用于人类或动物卵巢组织，以评估卵泡在发育各个阶段的基因表达谱10，11，12。然而，最佳分离程序应考虑到致密皮层内卵泡的脆弱结构，因此应小心执行以避免任何损害7。这份手稿描述了一种程序，改编自Woodruff实验室描述的方案，从冷冻解冻的卵巢皮层中分离出人类卵泡，以便进行基因表达分析13。
从冷冻人体组织中分离卵巢卵泡的第一步是解冻程序。该过程基于用于移植冷冻保存的卵巢组织的临床方案进行，如前所述14，15。该过程旨在通过以降低培养基浓度冲洗卵巢皮层来去除冷冻保护剂。然后，在酶和机械分离之前将组织碎片化以取回卵泡。可以使用具有高放大倍率和高质量光学元件的体视显微镜区分不同阶段的卵泡，以分离感兴趣的卵泡。使用集成到显微镜中的尺子测量每个分离的卵泡，并且可以根据卵泡的发育阶段合并卵泡：原始卵泡（30μm），初级卵泡（60μm），次级卵泡（120-200μm）和窦卵泡（>200μm）16。可以根据卵泡的形态进行进一步的分类：原始卵泡有一层扁平的颗粒细胞（GC），原代卵泡有一层立方体GC，次级卵泡至少有两层立方体GC，GC中存在空腔是窦期的特征。当选择感兴趣的卵泡时，进行RNA提取。在实时定量聚合酶链反应（RT-qPCR）之前评估RNA的数量和质量（图1）。
Protocol
该项目得到了伊拉斯姆医院伦理委员会（比利时布鲁塞尔）的批准。该方案中包含的患者在2000年化疗暴露之前接受了卵巢组织冷冻保存（OTC）以保存生育能力。患者签署了知情书面同意书，在储存期结束时将其剩余的冷冻组织捐赠给研究。 1. 冷冻保存的卵巢组织解冻 准备含有五种解冻溶液的 6 孔板。第一个孔含有 5 mL 由 Leibovitz-15 培养基、0.1 mol/L 蔗糖、…
Representative Results
使用此分离程序，实验者可以从基质环境中检索卵泡以进行特定的基因表达分析。根据卵泡的大小和形态，可以区分卵泡生成的不同阶段。实验者可以使用适应的微毛细管移液管根据卵泡的大小选择感兴趣的卵泡。通过使用最大75μm的微毛细血管，可以将原始和初级卵泡与次级，窦和成熟卵泡区分开来。此外，实验者可以根据卵泡形态确认卵泡阶段。在研究卵泡活化时，选择静止和原代卵泡，大?…
Discussion
卵巢组织的冷冻保存是保存癌症患者生育能力的一种有前途的方法。在临床上，解冻的皮质组织在缓解后移植回患者体内，允许卵巢功能和生育能力恢复19，20。除了临床使用外，残留的卵巢碎片也可以在储存期结束时捐赠用于研究，以研究调节卵泡生成的机制。此外，当不可行时，这种组织对于开发移植的替代方案特别有用，例如 体外 培养系?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了卓越科学（EOS）资助（ID：30443682）的支持。I.D.是比利时国家科学研究基金会（FNRS）的副研究员。
Materials
| 2 mm gridded Petri dish | Corning | 430196 | |
| 2100 Bioanalyzer instrument | Agilent | G2939BA | |
| 2100 Expert software | Agilent | version B.02.08.SI648 | |
| 4-wells plate | Sigma Aldrich | D6789 | |
| 6-wells plate | Carl Roth | EKX5.1 | |
| Agilent total RNA 6000 pico kit | Agilent | 5067-1513 | |
| Ascorbic acid | Sigma Aldrich | A4403 | |
| Aspirator tube assemblies for microcapillary pipettes | Sigma Aldrich | A5177 | |
| Centrifuge | Eppendorf | 5424R | |
| Collagenase IV | LifeTechnologies | 17104-019 | |
| DMSO | Sigma Aldrich | D2650 | |
| DNase | Sigma Aldrich | D4527-10kU | |
| FBS | Gibco | 10270-106 | |
| GoScript reverse transcriptase | Promega | A5003 | |
| HSA | CAF DCF | LC4403-41-080 | |
| Leibovitz-15 | LifeTechnologies | 11415-049 | |
| L-Glutamine | Sigma Aldrich | G7513 | |
| McCoy’s 5A + bicarbonate + Hepes | LifeTechnologies | 12330-031 | |
| McIlwain tissue chopper | Stoelting | 51350 | |
| Microcapillary RI EZ-Tips 200 µm | CooperSurgical | 7-72-2200/1 | |
| Microcapillary RI EZ-Tips 75 µm | CooperSurgical | 7-72-2075/1 | |
| NanoDrop 2000/2000c operating software | ThermoFisher | version 1.6 | |
| NanoDrop spectrophotometer | ThermoFisher | 2000/2000c | |
| Penicillin G | Sigma Aldrich | P3032 | |
| PowerTrack SYBR green master mix | ThermoFisher | A46109 | |
| Primers: GDF9 | F: CCAGGTAACAGGAATCCTTC R: GGCTCCTTTATCATTAGATTG | ||
| Primers: HPRT | F: CCTGGCGTCGTGATTAGTGAT R: GAGCACACAGAGGGCTACAA | ||
| Primers: Kit Ligand | F: TGTTACTTTCGTACATTGGCTGG R: AGTCCTGCTCCATGCAAGTT | ||
| Real-Time qPCR Quantstudio 3 | ThermoFisher | A33779 | |
| RNAqueous-micro total RNA isolation kit | ThermoFisher | AM1931 | |
| Selenium | Sigma Aldrich | S9133 | |
| Sodium pyruvate | Sigma Aldrich | S8636 | |
| Stereomicroscope | Nikon | SMZ800 | |
| Streptomycine sulfate | Sigma Aldrich | S1277 | |
| Sucrose | Sigma Aldrich | S1888 | |
| Thermo Scientific Forma Series II water-jacketed CO2 incubators | ThermoFisher | 3110 | |
| Thomas Stadie-Riggs tissue slicer | Thomas Scientific | 6727C10 | |
| Transferrin | Roche | 10652202001 |
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Cite This Article
Devos, M., Dias Nunes, J., Demeestere, I. Gene Expression Analyses in Human Follicles. J. Vis. Exp. (192), e64807, doi:10.3791/64807 (2023).