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Biologia

使用 体内 抑制、免疫荧光和流式细胞术对精子细胞中驱动蛋白-7 CENP-E 进行功能评估

Published: December 28, 2021
doi:
10.3791/63271
, , , , , , ,
1Department of Cell Biology and Genetics, The School of Basic Medical Sciences,Fujian Medical University, 2Key Laboratory of Stem Cell Engineering and Regenerative Medicine,Fujian Province University, 3Fujian Obstetrics and Gynecology Hospital, 4Medical Research Center, Fujian Maternity and Child Health Hospital,Affiliated Hospital of Fujian Medical University

Summary

本文报道了通过腹部手术和睾丸注射GSK923295对CENP-E的 体内 抑制，这是男性减数分裂的宝贵模型。使用免疫荧光，流式细胞术和透射电子显微镜测定，我们发现CENP-E抑制导致小鼠精子细胞中的染色体错位和基因组不稳定。

Abstract

在真核生物中，减数分裂对于有性生殖的基因组稳定性和遗传多样性至关重要。睾丸中精子细胞的实验分析对于研究男性减数分裂中的纺锤体组装和染色体分离至关重要。小鼠精子细胞是减数分裂机制研究的理想模型，然而，缺乏分析精子细胞的有效方法。本文报道了一种实用有效的小鼠精子细胞体内抑制驱动蛋白-7 CENP-E的方法。介绍了在3周龄小鼠中通过腹部手术GSK923295睾丸注射特定抑制剂的详细程序。此外，这里描述的是一系列用于组织收集和固定、苏木精-伊红染色、免疫荧光、流式细胞术和透射电子显微镜的方案。在这里，我们提出了一种通过腹部手术和睾丸注射的体内抑制模型，这可能是研究男性减数分裂的强大技术。我们还证明，在减数分裂I期间，CENP-E抑制导致原代精细胞的染色体错位和中期停滞。我们的体内抑制方法将促进减数分裂的机制研究，作为雄性生殖系遗传修饰的有用方法，并为未来的临床应用提供启示。

Introduction

减数分裂是真核生物中最重要、高度刚性、进化保守的事件之一，对配子发生、有性生殖、基因组完整性和遗传多样性至关重要1,2,3。在哺乳动物中，生殖细胞在单轮DNA复制后经历两次连续的细胞分裂，即减数分裂I和II。与有丝分裂中的姐妹染色单体不同，重复的同源染色体在减数分裂I 4,5期间配对并分离成两个子细胞。在减数分裂II中，姐妹染色单体拉开并分离形成单倍体配子，没有DNA复制6。两个减数分裂中的任何一个的错误，包括纺锤体组装缺陷和染色体偏置，都可能导致配子丢失、不育或非整倍体综合征 7,8,9。

越来越多的研究表明，驱动蛋白家族马达在有丝分裂和减数分裂细胞的染色体排列和分离、纺锤体组装、细胞分裂和细胞周期进展的调节中起着至关重要的作用10,11,12。Kinesin-7 CENP-E（着丝粒蛋白E）是染色体分裂13,14,15,16,17,18中染色体聚集，染色体运输和对齐以及纺锤体组装检查点调节所需的正端导向动粒马达。在减数分裂过程中，特异性抑制剂GSK923295的CENP-E抑制导致生精细胞的细胞周期停滞，染色体错位，纺锤体紊乱和基因组不稳定19。CENP-E在分裂精子细胞着丝粒处的定位模式和动力学表明，在减数分裂I20,21期间，CENP-E与动粒蛋白相互作用，以实现着丝粒的顺序组装。在卵母细胞中，CENP-E是染色体排列和减数分裂I13,22,23完成所必需的。抗体或吗啉基注射CENP-E导致小鼠和果蝇卵母细胞中的染色体错位，运动体方向异常和减数分裂I停滞23。与CENP-E在有丝分裂中的重要作用相比，CENP-E在减数分裂中的功能和机制在很大程度上仍然未知。CENP-E在雄性减数分裂细胞染色体聚集和基因组稳定性中的详细机制仍有待阐明。

精子发生是一个复杂而持久的生理过程，涉及顺序精子增殖、减数分裂和精子发生。因此，整个过程在哺乳动物和其他物种中体外复制是非常困难的24,25。在体外厚皮期后诱导精子细胞分化是不可能的。对男性减数分裂的研究一般仅限于早期减数分裂前期25,26的实验分析。尽管有许多技术努力，包括精子细胞的短期培养27,28和器官培养方法25，但研究男性减数分裂的有效方法很少。此外，必需基因的基因缺失通常会导致发育停滞和胚胎致死。例如，缺乏CENP-E的小鼠胚胎无法植入并且不能发展过去植入29，这是CENP-E在减数分裂中的机制研究的障碍。综上所述，建立切实可行的男性减数分裂研究体系，可以极大地促进减数分裂的研究领域。

小细胞渗透性抑制剂是研究细胞分裂和发育过程中驱动蛋白马达的有力工具。变构抑制剂GSK923295特异性结合CENP-E运动域，阻断ADP（二磷酸腺苷）的释放，最终稳定CENP-E与微管之间的相互作用30。在这项研究中，通过腹部手术和睾丸注射GSK923295提出了体内抑制小鼠模型。CENP-E抑制导致原代精细胞中期I的染色体错位。此外，CENP-E抑制导致精子细胞的减数分裂停滞和精子发生的破坏。描述了一系列用于分析精子细胞的方案，可用于观察精子细胞中的减数分裂纺锤体微管、同源染色体和亚细胞器。我们的体内抑制法是研究减数分裂和精子发生的有效方法。

Protocol

所有动物实验均由福建医科大学动物护理与使用委员会审查和批准（实验方案编号SYXK 2016-0007）。所有小鼠实验均按照美国国立卫生研究院实验动物护理和使用的相关指南（NIH出版物编号8023,1978年修订）进行。 1. GSK923295介导的CENP-E抑制小鼠模型的构建 在121°C下对手术器械进行灭菌30分钟。用紫外线C（UVC）照射手术超净工作台2小时。称量用于实验的3周龄雄…

Representative Results

通过腹部手术和睾丸注射GSK923295 19，成功构建了小鼠睾丸体内CENP-E抑制模型。该方法的关键技术步骤如图1所示。睾丸注射GSK923295 4天后，收获睾丸进行进一步分析。在对照组中，生精小管中的生精波是规则且有组织的（图2A）。然而，在GSK923295组中，生精波在生精小管中发生改变，并且在CENP-E抑制后中期停滞的原代精子细胞显?…

Discussion

在这项研究中，我们利用腹部手术和显微注射GSK923295建立了小鼠睾丸体内CENP-E抑制模型。本研究中使用的腹部手术和睾丸注射方法具有以下优点。首先，它不仅限于小鼠的年龄。实验者可以在早期阶段进行睾丸注射，例如，在3周龄或更年轻的小鼠身上。其次，GSK923295对CENP-E具有特异性和优异的抑制作用。第三，该方法操作简单，可重复性高。此外，睾丸的完整性得以保持，这适用于在器官…

Declarações

The authors have nothing to disclose.

Acknowledgements

我们感谢福建医科大学细胞骨架实验室的所有成员的有益讨论。感谢福建医科大学公共技术服务中心林俊进在流式细胞术方面的技术支持。感谢福建医科大学公共技术服务中心电子显微镜实验室的吴明霞和周林英在电子显微镜方面的技术援助。感谢福建医科大学基础医学实验教学中心的郑思义、林颖、祁珂、宋军的支持。本研究得到了以下资助：国家自然科学基金（批准号82001608），中国福建省自然科学基金（批准号2019J05071），福建省卫生技术项目（批准号2018-1-69），福建医科大学科研启动基金（批准号2017XQ1001），福建医科大学高层次人才科研启动资金项目（批准号XRCZX2017025）和研究项目中医研究生网上教育与教学（资助号B-YXC20200202-06）。

Materials

0.25% Trypsin-EDTA Gibco 25200056
1 ml Syringe Several commercial brands available Sterile.
1.5 mL Centrifuge tube Axygen MCT-150-C
50 mL Centrifuge Tube Corning 430828
6 cm Petri dish Corning 430166
95% Ethanol Sinopharm Chemical Reagent Co.,Ltd 10009164
tubulin rabbit polyclonal antibody Beyotime AF0001 For immunofluorescence assays. Use at 1:100.
rabbit anti-Histone H3 (phospho S10) monoclonal antibody Abcam ab267372 For immunofluorescence assays. Use at 1:100.
rabbit anti-TUBA4A polyclonal antibody Sangon Biotech D110022 For immunofluorescence assays. Use at 1:100.
Anti-SYCP3 rabbit monoclonal antibody Abcam ab175191 For immunofluorescence assays. Use at 1:100.
Adhesion microscope slides CITOTEST 188105
Alexa fluor 488-labeled goat anti-rabbit antibody Beyotime A0423 Sencodary antibody. Use at 1:500.
Aluminium potassium sulphate Sinopharm Chemical Reagent Co.,Ltd 10001060
Anhydrous ethanol Sinopharm Chemical Reagent Co.,Ltd 100092690
Anti-fade mounting medium Beyotime P0131 Prevent photobleching of flourescent signals.
BD FACS Canto II BD Biosciences FACS Canto II
Bovine Serum Albumin Sinopharm Chemical Reagent Co.,Ltd 69003435
Centrifuge Eppendorf 5424BK745380
Chloral hydrate Sinopharm Chemical Reagent Co.,Ltd 80037516
Citric acid Shanghai Experiment Reagent Co., Ltd 122670
Collagenase Sangon Biotech A004194-0100
Coverslips CITOTEST 10212020C 20 × 20 mm. Thickness 0.13-0.16 mm.
DAPI Beyotime C1006
Dye vat Several commercial brands available 91347802
Eosin Y, alcohol soluble Sinopharm Chemical Reagent Co.,Ltd 71014460
Ether Sinopharm Chemical Reagent Co.,Ltd 10009318
Formaldehyde – aqueous solution Sinopharm Chemical Reagent Co.,Ltd 10010018
GSK923295 MedChemExpress HY-10299
Hematoxylin, anhydrous Sinopharm Chemical Reagent Co.,Ltd 71020784
ICR mouse Shanghai SLAC Laboratory Animal Co., Ltd
Image J software National Institutes of Health https://imagej.nih.gov/ij/ Fluorescent image analysis.
Leica ultramicrotome Leica
Leica EM UC-7 ultramicrotome Leica EM UC7
Modfit MFLT32 Verity Software House For analysis of flow cytometry results.
Nail polish Several commercial brands available
Neutral gum Sinopharm Chemical Reagent Co.,Ltd 10004160
Nikon Ti-S2 microscope Nikon Ti-S2
Picric acid Sinopharm Chemical Reagent Co.,Ltd J60807
Rheodyne Sangon Biotech F519160-0001 10 μl rheodyne
Sliced paraffin Sinopharm Chemical Reagent Co.,Ltd 69019461
Sodium iodate Sinopharm Chemical Reagent Co.,Ltd 80117214
Surgical instruments Several commercial brands available For abdominal surgery. Sterilize at 121 °C, 20 min.
Transmission electron microscope FEI Tecnai G2
Trisodium citrate dihydrate Shanghai Experiment Reagent Co., Ltd 173970
Triton X-100 Sinopharm Chemical Reagent Co.,Ltd 30188928 Dilute in sterile PBS to make a 0.25% working solution.
Tween 20 Sinopharm Chemical Reagent Co.,Ltd 30189328 Dilute in sterile PBS to make a 0.1% working solution.
Paraformaldehyde Sinopharm Chemical Reagent Co.,Ltd 80096618
Xylene Sinopharm Chemical Reagent Co.,Ltd 10023418
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Tags

Keywords: MeiosisSpermatogenesisSpermatocytesCENP-EGSK923295ImmunofluorescenceFlow CytometryGene EditingMouse ModelMicrotome
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Xu, M., Yang, Y., Wei, Y., Zhang, J., Lin, X., Lin, X., Chen, H., She, Z. Functional Assessment of Kinesin-7 CENP-E in Spermatocytes Using In Vivo Inhibition, Immunofluorescence and Flow Cytometry. J. Vis. Exp. (178), e63271, doi:10.3791/63271 (2021).
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