附睾蛋白的合成与分泌分析
1Priority Research Centre for Reproductive Science, School of Environmental and Life Sciences,University of Newcastle, 2Hunter Medical Research Institute, 3Department of Physiology, Turku Center for Disease Modeling, Institute of Biomedicine,University of Turku, 4School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine,University of Newcastle
Summary
在这里, 我们报告动力蛋白的免疫荧光定位, 以说明在石蜡嵌入小鼠附睾切片和永生化附睾细胞系 (mECap18) 的蛋白质检测协议。我们还描述了从附睾液和条件细胞培养基中分离分泌蛋白的协议。
Abstract
哺乳动物附睾生成任何内分泌腺体中最复杂的腔内液体之一, 以支持睾丸后成熟和精子的贮存。这种复杂性的产生是由于衬里上皮细胞的分泌和吸收活性的综合作用。在这里, 我们描述的技术, 分析附睾蛋白合成和分泌的重点是模型蛋白家族的动力蛋白 (DNM) mechanoenzymes;具有调节双向膜贩运活动潜力的大型 GTPases。为了研究附睾组织中蛋白质的表达, 我们描述了石蜡嵌入切片中靶蛋白免疫荧光标记的鲁棒方法, 并通过对这些蛋白质的空间分布进行了检测。免疫荧光显微镜。我们还描述了最优化的方法, 以分离和鉴定的外切像囊泡, 称为 epididymosomes, 这是分泌到附睾腔参与细胞间沟通与成熟的精子细胞。作为补充方法, 我们还描述了免疫荧光检测的目标蛋白在 SV40-immortalized 小鼠附睾上皮 (mECap18) 细胞系。此外, 我们还讨论了 mECap18 细胞系作为一种合适的体外模型, 用于研究附睾分泌活动的调节。为此, 我们描述了维持 mECap18 细胞系的培养要求和使用有选择性的药理抑制方案, 它们能够影响其分泌蛋白的分布。后者很容易通过收获的条件培养基, 分泌蛋白的浓度通过乙酸酸/丙酮沉淀和他们随后的分析通过 SDS 页和免疫印迹法。我们认为, 这些联合方法适合于分析替代附睾蛋白靶点, 作为确定它们在精子成熟和/或贮存中功能作用的前奏。
Introduction
所有哺乳动物的精子都有可能显示正向渐进运动, 并通过附睾 (雄性额外睾丸导管系统的高度专门化区域) 受精卵子, 这可能用 7-14 天的时间来导航 (取决于物种)1。由于父亲染色质的极度凝结和大部分细胞质的脱落, 伴随着睾丸内精子的 cytodifferentiation, 它们随后的功能成熟完全由它们的相互作用驱动。附睾微环境。这一环境反过来, 由内衬附睾的分泌和吸收活动创造, 并显示一个特殊水平的段段变化1。因此, 在蛋白质合成和分泌方面, 最活跃的部分是位于附睾近端的部分 (即, 额和语料库)2。这项活动反映精子的功能轮廓, 细胞首先开始显示功能能力的特征 (i. e., 渐进动力和绑定到酸可溶性透明糖蛋白的能力) 后, 其通过3。这些功能属性继续发展之前达到最佳水平, 因为精子到达远端附睾段 (马尾), 其中他们储存在一个静止状态, 准备射精。这种精子储存库的形成和维持也与衬里上皮紧密相连, 在马尾中由强吸收活性4、5控制。虽然解剖差异已报告6,7,8, 这种分区分工似乎是一个特点的附睾, 是共享的大多数哺乳动物物种研究至今, 包括我们自己的9,10。事实上, 从临床的角度来看, 附睾功能障碍对男性因素不孕11的病因有重要的贡献, 因此突出了了解这种特殊组织的调节的重要性。
因此, 令人遗憾的是, 我们对附睾生理的理解, 以及调控精子成熟和贮存的顺序阶段的机制, 仍有待完全解决。在促成因素中, 附睾研究的进展受到限制, 这种组织的总体复杂性和对其管腔微环境的调控机制的认识。解剖上, 我们知道, 除了额位, 语料库和马尾段的区别, 附睾可以进一步细分为几个区域 (图 1A), 每隔12间隔, 并以基因/蛋白的离散剖面为特征表达式13,14,15,16,17,18。事实上, 根据对附睾节段基因表达的详细转录分析, 老鼠和大鼠模型中有多达6和9个不同的附睾区, 分别为19、20。这种复杂性大概反映了附睾躯体的组成, 假上皮包括许多不同的细胞类型;每种不同的, 他们的丰度, 分布和分泌/吸收活动的长度。因此, 主细胞是迄今为止最丰富的附睾细胞类型, 构成了所有上皮细胞的80% 以上。因此, 主要细胞负责附睾蛋白生物合成和分泌物的大部分5。与此相反, 作为附睾躯体内第二大细胞类型的透明细胞种群, 主要参与腔内成分的选择性吸收和这种微环境的酸化5。添加另一层复杂性, 雄激素和其他 lumicrine 因素的睾丸起源施加差异控制每一个这些附睾细胞类型取决于他们的定位沿道。
尽管这种复杂性所造成的限制, 仍有重大进展, 以解决附睾功能的机制基础。这些研究的一个关键是应用先进的质谱策略建立广泛的附睾蛋白质组的清单, 并与从这些初步调查中选择的单个蛋白质进行详细分析。这一方法的一个例证是我们最近在老鼠模型21中对 mechanoenzymes DNM 家族的描述。我们最初对 DNM 的兴趣是由它在 endocytotic 过程耦合中的双重作用所推动的。在这些观察的基础上, 我们能够证明 DNM (DNM1-DNM3) 的三规范亚型在小鼠附睾中有高度表达, 并适当定位以在蛋白质分泌和吸收中发挥调节作用21.此外, 我们能够清楚地区分每个 DNM 异构体的基础上, 他们的细胞和亚细胞定位, 从而表明, 他们拥有互补, 而不是多余的活动, 在附睾上皮21。
在这里, 我们描述了用于研究小鼠附睾中 DNM 表达的实验方法, 希望这些信息能在其他附睾蛋白的鉴定中得到更广泛的应用, 从而有助于我们了解男性生殖道这一重要元素的功能。具体来说, 我们描述了在石蜡嵌入附睾切片中靶蛋白免疫荧光标记的稳健方法的发展, 以及随后通过免疫荧光检测这些蛋白质的空间分布。显微镜。我们进一步记录我们最近优化的协议22 , 以隔离和表征 epididymosomes;小的外切状囊泡, 构成附睾分泌剖面的关键元素, 在促进精子成熟23中似乎起着突出的作用。作为补充的方法, 我们还描述了免疫荧光检测的目标蛋白在永生化鼠额附睾上皮 (mECap18) 细胞系和利用这个资源作为一个模型, 以探讨对附睾的调节体外分泌活性。
Protocol
所有涉及动物组织收集的实验程序都是由纽卡斯尔大学动物保育和伦理委员会批准的。 1. 石蜡嵌入附睾切片的免疫荧光染色 (图1和 2) 在成年小鼠安乐死后, 通过 CO2吸入 (瑞士老鼠, 超过8周), 仔细解剖附睾 (使用手术剪刀和镊子) 没有覆盖结缔组织和脂肪, 浸泡在 Bouin 的固定器解决方案 (> 十倍体积/组织重量) 过夜固定。 每天用70% 乙醇与2×改变2天, 然后通…
Representative Results
图 1和图 2显示了小鼠附睾 DNM 免疫荧光定位的代表性结果。调查的三 DNM 亚型中的每个都显示不同的定位配置文件。因此, DNM1 的特点是相对温和的弥漫性标记的附睾细胞在整个初始段和额附睾 (图 2A)。相比之下, DNM2 异构体首先检测到在最初的部分细胞的对立的基底和顶端边界附近, 然后被重?…
Discussion
这些研究纳入了使用 Bouin 的固定附睾组织, 已受到石蜡嵌入和标准切片协议。Bouin 的固定液包括甲醛、苦味酸酸和乙酸的混合物, 每个组分具有特定的互补作用。因此, 甲醛与原胺反应形成蛋白交联, 苦味酸酸慢慢穿透组织形成的盐, 从而使碱性蛋白凝固, 反之, 醋酸迅速穿透组织, 导致凝血核酸。这些组合的性质产生了 Bouin 作为一个固定的选择保存的形态学细节, 它的使用被广泛报道的附睾文献。?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
提交人要感谢澳大利亚国家卫生和医学研究理事会项目赠款 APP1103176 支持这项工作。
Materials
| Dynamin 1 antibody | Abcam | ab108458 | Host species: Rabbit, Isotype: IgG, Class: polyclonal |
| Dynamin 2 antibody | Santa Cruz | sc-6400 | Host species: Goat, Isotype: IgG, Class: polyclonal |
| Dynamin 3 antibody | Proteintech | 14737-1-AP | Host species: Rabbit, Isotype: IgG, Class: polyclonal |
| ATP6V1B1 antibody | Santa Cruz | sc-21206 | Host species: Goat, Isotype: IgG, Class: polyclonal |
| CD9 antibody | BD Pharmingen | 553758 | Host species: Rat, Isotype: IgG, Class: monoclonal |
| Flotillin-1 antibody | Sigma | F1180 | Host species: Rabbit, Isotype: IgG, Class: polyclonal |
| ALOX15 antibody | Abcam | ab80221 | Host species: Rabbit, Isotype: IgG, Class: polyclonal |
| TUBB antibody | Santa Cruz | sc-5274 | Host species: Mouse, Isotype: IgG, Class: monoclonal |
| PSMD7 antibody | Abcam | ab11436 | Host species: Rabbit, Isotype: IgG, Class: polyclonal |
| Anti Rabbit Alexa Fluor 488 | Thermo | A11008 | Host species: Goat, Isotype: IgG, Class: polyclonal |
| Anti Goat Alexa Fluor 488 | Thermo | A11055 | Host species: Donkey, Isotype: IgG, Class: polyclonal |
| Anti Goat Alexa Fluor 594 | Thermo | A11058 | Host species: Donkey, Isotype: IgG, Class: polyclonal |
| Anti Rat Alexa Fluor 594 | Thermo | A11007 | Host species: Goat, Isotype: IgG, Class: polyclonal |
| Anti Rabbit HRP | Millipore | DC03L | Host species: Goat, Isotype: IgG, Class: polyclonal |
| Anti Rat HRP | Millipore | DC01L | Host species: Goat, Isotype: IgG, Class: polyclonal |
| Anti Mouse HRP | Santa Cruz | sc-2005 | Host species: Goat, Isotype: IgG, Class: polyclonal |
| 4', 6-diamidino-2-phenylindole (DAPI) | Sigma | D9564 | |
| propidium iodide (PI) | Sigma | P4170 | |
| Mowiol 4-88 | Calbiochem | 475904 | |
| Bovine serum albumin (BSA) | Sigma | A7906 | |
| fetal bovine serum (FBS) | Bovogen | SFBS-F | |
| DMEM | Thermo | 11960-044 | |
| L-glutamine | Thermo | 25030-081 | |
| penicillin/streptomycin | Thermo | 15140-122 | |
| 5α-androstan-17β-ol-3-oneC-IIIN | Sigma | A8380 | |
| sodium pyruvate | Thermo | 11360-070 | |
| Trypsin-ethylenediaminetetraacetic acid (EDTA) | Sigma | T4049 | |
| Paraformaldehyde (PFA) | EMS | 15710 | |
| Xylene | VWR Chemicals | 1330-20-7 | |
| Ethanol | VWR Chemicals | 64-17-5 | |
| Phosphate buffered saline (PBS) | Sigma | P4417 | |
| Sodium citrate | Sigma | S1804 | |
| Tris | Astral | 0497-5KG | |
| Glycerol | Sigma | G5516 | |
| 1, 4-diazabicyclo-(2.2.2)-octane | Sigma | D2522 | |
| Poly-L-gysine | Sigma | P4832 | |
| Triton X-100 | Sigma | 78787 | |
| Trypan blue | Sigma | T6146 | |
| Trichloroacetic acid | Sigma | T9159 | |
| Acetone | Ajax Finechem | A6-2.5 L GL | |
| Sucrose | Sigma | S0389 | |
| Poly (vinyl alcohol) | Sigma | P8136 | |
| D-Glucose | Ajax Finechem | 783-500G | |
| OptiPrep Density Gradient Medium | Sigma | D1556 | |
| Fluorescence microscopy | Zeiss | Zeiss Axio Imager A1 | |
| Ultracentrifuge | BECKMAN COULTER | Optima Max-XP | |
| Microcentrifuges | Eppendorf | 5424R | |
| Incubator | Heracell | 150 | |
| Large Orbital Shaker | Ratek | OM7 | |
| Microwave | LG | MS3840SR /00 | |
| Lab pH Meter | MeterLab | PHM220 | |
| Liquid-repellent slide marker | Daido Sangyo | Mini | |
| Coverslip | Thermo | 586 | |
| 6 well plate | CELLSTAR | 657160 | |
| 12 well plate | CELLSTAR | 665180 | |
| Slide | Mikro-Glass | SF41296PLMK | |
| 0.45 µm filter | Millox-HV | SLHV033RS | |
| Kimwipes Dustfree Paper | KIMTECH | 34155 | |
| Ultracentrifuge tube (2.2 ml, 11 × 35 mm) | BECKMAN COULTER | 347356 | |
| Ultracentrifuge tube (3.2 ml, 13 × 56 mm) | BECKMAN COULTER | 362305 | |
| Cell strainer 70 µm Nylon | FALCON | 352350 | |
| Petri dish 35 × 10 mm with cams | SARSTED | 82.1135.500 | |
| Slide jar | TRAJAN | #23 319 00 |
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Citazione di questo articolo
Zhou, W., Sipilä, P., De Iuliis, G. N., Dun, M. D., Nixon, B. Analysis of Epididymal Protein Synthesis and Secretion. J. Vis. Exp. (138), e58308, doi:10.3791/58308 (2018).