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
Summary
推进前窦卵泡生成的研究需要从单个卵巢中分离卵泡的有效方法。这里介绍的是一种简化的机械方案，用于使用组织切碎器和均质器从牛卵巢中分离卵泡。该方法允许从单个卵巢中收集大量活的前窦卵泡。
Abstract
了解哺乳动物卵泡发生的完整过程对于改善牲畜、人类和濒危物种的辅助生殖技术至关重要。由于难以分离较小的前窦卵泡，特别是在牛等大型哺乳动物中，研究主要局限于窦前卵泡和大前窦卵泡。这项工作提供了一种从单个牛卵巢中取出大量小前窦卵泡的有效方法。使用组织切碎器将单个牛卵巢的皮层切成500μm立方体，并使用10mm探针以9，000-11，000rpm匀浆6分钟。使用奶酪布从匀浆中分离大碎片，然后通过300μm和40μm细胞过滤器进行连续过滤。将保留在40μm过滤器中的内容物冲洗到搜索皿中，在那里鉴定卵泡并将其收集到一滴培养基中。通过台盼蓝染色 测试 收集的卵泡的活力。该方法能够在大约90分钟内从单个牛卵巢中分离出大量活的小前窦卵泡。重要的是，这种方法完全是机械的，避免使用酶解离组织，这可能会损害卵泡。使用该协议获得的卵泡可用于下游应用，例如用于RT-qPCR的RNA分离，特定蛋白质的免疫定位和 体外 培养。
Introduction
卵巢卵泡是卵巢的功能单位，负责配子（卵母细胞）的产生以及对生殖功能和整体健康至关重要的激素。原始卵泡在胎儿发育期间或新生儿期在卵巢中形成，具体取决于物种1，它们构成了女性的卵巢储备。卵泡生长始于原始卵泡的激活，这些卵泡离开休息池并进入生长阶段。前窦卵泡发生，包括窦发育前的所有卵泡阶段，是一个高度动态的过程，需要卵母细胞和周围颗粒细胞的同步形态和代谢变化，由这两种细胞类型之间的紧密通信驱动2，3。前窦卵泡构成了在任何给定时间在卵巢中发现的大部分滤泡单位4.据估计，通过卵泡生成的前窦阶段的发育比窦发育长数周5，6，并且这段时间是卵母细胞和体细胞获得足够成熟以进入发育的最后阶段（即窦期）所必需的，并为排卵，受精和胚胎发育做准备7，8，9。
目前关于卵巢前窦卵泡发生的大部分知识来自小鼠模型10，11，12，13，部分原因是从较小且纤维较少的卵巢中恢复大量这些卵泡的容易程度。尽管从牛卵巢中分离出大量前窦卵泡的报道可以追溯到大约30年前14，但对这些早期卵泡发育的调节过程的更完整的理解仍未实现，主要是由于缺乏优化，有效和可重复的方法来检索足够数量的活前窦卵泡，特别是在发育的早期阶段。随着人们对保存卵巢储备以备将来用于人类辅助生殖的兴趣日益浓厚，奶牛因其更相似的卵巢结构而成为有吸引力的模型15。然而，与小鼠卵巢16相比，牛卵巢的胶原蛋白明显更丰富，使得使用为小鼠描述的方法进行机械隔离的效率非常低。扩大生育力保存技术的努力包括将前窦卵泡完全体外生长到窦期，然后是封闭卵母细胞的体外成熟（IVM），体外受精（IVF）以及胚胎的产生和移植17。到目前为止，整个过程仅在小鼠18中实现。在牛中，体外卵泡生长的进展仅限于培养开始时具有可变卵泡阶段的少数报告，以及方案17，19之间的可变培养长度。
文献中描述的从牛卵巢中收获前窦卵泡的方法大多使用机械和酶促技术，无论是分离的还是组合的2，14，17，20。牛前窦卵泡分离方案的第一份报告使用组织匀浆器和连续过滤来处理整个卵巢20。这项研究之后，报告了利用胶原酶14的机械和酶程序。当利用胶原酶消化卵巢组织时，一个反复出现的主题是滤泡基底膜受损的潜在风险，这可能会损害卵泡活力14，21，22，23。因此，已经采用了不同的机械方法组合，例如使用组织切碎器和重复移液或组织切碎器与均质化相结合20，24，25，26。已经描述的另一种机械技术利用针直接从卵巢组织中解剖前窦卵泡，这对于分离较大的（>200μm）次级卵泡特别有用。然而，这个过程是耗时的，对于分离较小的前窦卵泡效率低下，并且在牛卵巢中尝试时依赖于技能组19，27，28。
利用文献中描述的不同技术，该协议旨在以简单，一致和有效的方式优化单个牛卵巢中前窦卵泡的分离，避免在酶溶液中孵育。改进分离前窦卵泡的方法将为增强对这一卵泡生成阶段的理解提供机会，并使开发有效的培养系统以将前窦卵泡发育到窦期。本文描述的从大型哺乳动物（如牛种）中分离前窦卵泡的详细程序对于旨在研究可转化为人类的非小鼠物种的早期卵泡发生的研究人员至关重要。
Protocol
牛（Bos taurus）卵巢来自当地的屠宰场，并在收集后6小时内运送到实验室。由于该设施处理的动物数量众多，动物的年龄、品种和发情周期的阶段是未知的。由于这些实验中没有使用活体动物，因此不需要批准的动物护理和使用方案。 1. 设备和试剂的准备 用台纸覆盖实验室工作台的 2 英尺宽部分。 获得手术刀手柄，无菌手术刀刀片，止血器…
Representative Results
概述和关键步骤使用该协议，可以可靠地从单个卵巢中分离出实验相关数量的小牛前窦卵泡。从总共30个重复中，每个重复平均获得41个卵泡，范围为11至135个卵泡（图4A）。在14个重复中，通过在体视显微镜下使用1μm显微镜校准载玻片测量卵泡直径，如前所述26 所示，对卵泡发育阶段进行表征。使用这种方法，共有476个卵泡被分类为原发性?…
Discussion
本协议详细介绍了一种可重复的方法，用于从牛卵巢中取出早期前窦卵泡，特别是在初级和早期二级。该协议建立在先前的报告20，25，30，34，35，36的基础上，并提供优化，导致从单个卵巢中分离出有意义数量的卵泡。使用这种方法分离的前窦卵泡…
Declarações
The authors have nothing to disclose.
Acknowledgements
该项目部分由美国农业部多州项目W4112和加州大学戴维斯分校Jastro Shields授予SM资助。
作者要感谢Central Valley Meat，Inc.提供所有实验中使用的牛卵巢。作者还感谢Olivia Silvera在卵巢处理和卵泡分离方面的帮助。
Materials
| 5-3/4" Soda Lime Disposable Glass Pasteur Pipette | Duran Wheaton Kimble | 63A54 | Pasteur pipette that can be used to dislodge follicles from debris while searching within the petri dish |
| 16% Paraformaldehyde | Electron Microscopy Sciences | 15710 | Diluted to 4%; fixation of follicles for immunostaining |
| 20 mL Luer-lock Syringe | Fisher Scientific | Z116882-100EA | Syringe used with the 18 G needle to dislodge follicles from the 40 μm cell strainer |
| #21 Sterile Scalpel Blade | Fisher Scientific | 50-365-023 | Used to cut the ovaries and remove the medula |
| 40 μm Cell Strainer | Fisher Scientific | 22-363-547 | Used to filter the filtrate from the 300 μm cell strainer |
| 104 mm Plastic Funnel | Fisher Scientific | 10-348C | Size can vary, but ensure the cheese cloth is cut appropriately and that the ovarian homogenate will not spill over |
| 300 μm Cell Strainer | pluriSelect | 43-50300-03 | Used to filter the filtrate from the cheese cloth |
| 500 mL Erlenmeyer Flask | Fisher Scientific | FB500500 | Funnel and flask used to catch filtrate from the cheese cloth |
| Air-Tite Sterile Needles 18 G | Thermo Fisher Scientific | 14-817-151 | 18 G offers enough pressure to dislodge follicles from the 40 μm cell strainer |
| Air-Tite Sterile Needles 27 G 13 mm | Fisher Scientific | 14-817-171 | Needles that can be used to manipulate any debris in which follicles are stuck |
| BD Hoechst 33342 Solution | Fisher Scientific | BDB561908 | Fluorescent DNA stain |
| Bovine Serum Albumin (BSA) | Sigma-Aldrich | A7030-100G | Component of follicle wash media |
| Cheese Cloth | Electron Microscopy Sciences | 71748-00 | First filtering step of the ovarian homogenate meant to remove large tissue debris |
| Classic Double Edge Safety Razor Blades | Wilkinson Sword | N/A | Razor blades that fit the best in the McIlwain Tissue Chopper and do not dull quickly |
| Donkey-Anti-Rabbit Secondary Antibody, Alexa Fluor 488 | Fisher Scientific | A-21206 | Secondary antibody for immunostaining |
| Eisco Latex Pipette Bulbs | Fisher Scientific | S29388 | Rubber bulb to use with Pasteur pipettes |
| HEPES Buffer | Sigma-Aldrich | H3375 | Component of follicle wash media |
| Homogenizer | VWR | 10032-336 | Homogenize the ovarian tissue to release follicles |
| ImageJ/Fiji | NIH | v2.3.1 | Software used for analysis of fluorescence-immunolocalization |
| McIlwain Tissue Chopper | Ted Pella | 10184 | Used to cut ovarian tissue small enough for homogenization |
| Microscope – Stereoscope | Olympus | SZX2-ILLT | Dissection microscope used for searching and harvesting follicles from the filtrate |
| Microscope – Inverted | Nikon | Diaphot 300 | Inverted microscope used for high magnification brightfield visualization of isolated follicles |
| Microscope – Inverted | ECHO | Revolve R4 | Inverted microscope used for high magnification brightfield and epifluorescence visualization of isolated follicles |
| Mineral Oil | Sigma-Aldrich | M8410-1L | Oil to cover the drops of follicle wash medium to prevent evaporation during searching |
| Non-essential Amino Acids (NEAA) | Gibco | 11140-050 | Component of follicle wash medium |
| Normal Donkey Serum | Jackson ImmunoResearch | 017-000-001 | Reagent for immunostaining blocking buffer |
| Nunc 4-well Dishes for IVF | Thermo Fisher Scientific | 144444 | 4-well dishes for follicle isolation and washing |
| Penicillin-Streptomycin Solution 100x | Gibco | 15-140-122 | Component of follicle wash medium |
| Petri Dish 60 mm OD x 13.7 mm | Ted Pella | 10184-04 | Petri dish that fits the best in the McIlwain Tissue Chopper |
| Phosphate Buffered Saline (PBS) | Fisher Scientific | BP665-1 | Washing buffer for ovaries and follicles |
| Plastic Cutting Board | Fisher Scientific | 09-002-24A | Cutting board of sufficient size to safely cut ovaries |
| Polyvinylpyrrolidone (PVP) | Fisher Scientific | BP431-100 | Addition of PVP (0.1% w/v) to PBS prevents follicles from sticking to the plate or each other |
| ProLong Gold Antifade Mountant | Thermo Fisher Scientific | P36930 | Mounting medium for fluorescently labeled cells or tissue |
| Qiagen RNeasy Micro Kit | Qiagen | 74004 | RNA column clean-up kit |
| R | The R Foundation | v4.1.2 | Statistical analysis software |
| Rabbit-Anti-Human Cx37/GJA4 Polyclonal Antibody | Abcam | ab181701 | Cx37 primary antibody for immunostaining |
| RevertAid RT Reverse Transcription Kit | Thermo Fisher Scientific | K1691 | cDNA synthesis kit |
| Rstudio | RStudio, PBC | v2021.09.2 | Statistical analysis software |
| Sodium Hydroxide Solution (1N/Certified) | Fisher Scientific | SS266-1 | Used to increase media pH to 7.6-7.8 |
| Sodium Pyruvate (NaPyr) | Gibco | 11360-070 | Component of follicle wash medium |
| Square Petri Dish 100 mm x 15 mm | Thermo Fisher Scientific | 60872-310 | Gridded petri dishes allow for more efficient identification of follicles |
| SsoAdvanced Universal SYBR Green Supermix | BioRad | 1725271 | Mastermix for PCR reaction |
| Steritop Threaded Bottle Top Filter | Sigma-Aldrich | S2GPT02RE | Used to sterilize follicle wash medium |
| SYBR-safe DNA gel stain | Thermo Fisher Scientific | S33102 | Staining to visual PCR products on agarose gel |
| TCM199 with Hank’s Salts | Gibco | 12-350-039 | Component of follicle wash medium |
| Triton X-100 | Fisher Scientific | BP151-100 | Detergent for immunostaining permeabilization buffer |
| Trizol reagent | Thermo Fisher Scientific | 15596026 | RNA isolation reagent |
| Trypan Blue Solution, 0.4% | Gibco | 15-250-061 | Used for testing viability of isolated follicles |
| Tween 20 | Detergent for immunostaining wash buffer | ||
| Warmer Plate Universal | WTA | 20931 | Warm plate to keep follicles at 38.5 °C while searching under the microscope |
| Wiretrol II Calibrated Micropipets | Drummond | 50002-005 | Glass micropipettes to manipulate follicles |
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Citar este artigo
McDonnell, S. P., Candelaria, J. I., Morton, A. J., Denicol, A. C. Isolation of Small Preantral Follicles from the Bovine Ovary Using a Combination of Fragmentation, Homogenization, and Serial Filtration. J. Vis. Exp. (187), e64423, doi:10.3791/64423 (2022).