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Microscopic Observation, Three-dimensional Reconstruction, and Volume Measurements of Sperm Nuclei
精子细胞核的显微观察、三维重建和体积测量   

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本实验方案简略版
Plant Physiology
Aug 2014

Abstract

Karyogamy, a migration of the sperm nucleus toward the egg nucleus and their subsequent nuclear fusion, is an important biological event for initiating zygote formation toward early embryogenesis in angiosperms. However, how the male nucleus approaches and fuses with the female nucleus still remains unclear. Recently, time-lapse measurement of nuclear volume during karyogamic events revealed that the sperm nucleus enlarges during contact with the egg nucleus via possible one-directional migration of egg chromatin into sperm nucleus (Ohnishi et al., 2014). Here, we describe the protocol for microscopic observation, three-dimensional reconstruction, and volume measurements of sperm nuclei in rice zygotes/fused gametes, which are produced by an in vitro fertilization system (Uchiumi et al., 2006; Uchiumi et al., 2007). The present protocol will be applied for monitoring nuclear dynamics in cells during cell division, differentiation, de-differentiation and polarity formation as well as karyogamy progression.

Keywords: Egg cell (卵细胞), Karyogamy (核融合), Nuclear fusion (核聚变), Sperm cell (精子细胞), Zygote (受精卵)

Materials and Reagents

  1. Transgenic rice plants (Oryza sativa L. cv. Nipponbare) expressing SUN2-GFP or H2B-RFP fusion proteins that are targeted to nuclear membrane or nuclear chromatin, respectively (Ohnishi et al., 2014) (see Note 1)
  2. Mannitol (Wako Chemicals USA, catalog number: 133-00845 )
  3. 370 mosmol/kg H2O (330 mM) mannitol solution (autoclaved)
  4. 450 mosmol/kg H2O (385 mM) mannitol solution (autoclaved)
  5. Mineral oil (Sigma-Aldrich, catalog number: M8410-100ML )

Equipment

  1. Inverted fluorescent microscope (OLYMPUS, model: BX-71 )
  2. Confocal laser scanning (CLS) microscope (ZEISS, model: LMS 710 )
  3. Non-treated plastic dishes with diameter of 3.5 cm (Iwaki, catalog number: 1000-035 )
  4. Glass base dishes with diameter of 3.5 cm (Iwaki, catalog number: 3971-035 ) (see Note 2)
  5. Coverslips (24 x 40 mm) (Thermo Fisher Scientific, catalog number: 125485J ) (siliconized at the edges with 5% dichloromethylsilane in 1,1,1-trichloroethane) (see Note 3)
  6. Glass capillaries made from 50 μl aspirator tubes (Drummond Scientific Company, catalog number: 2-000-050 ) (Figure 1A-B) (see Note 4)
  7. Manual handling injector (ST Science, type UJB)
  8. Environmental chamber (Koito Industries Ltd., catalog number: K30-7248 )

Software

  1. IMARIS software (Bitplane)
    Note: A core scientific software module that delivers the necessary functionality for data management, visualization, analysis, segmentation and interpretation of 3D and 4D microscopy datasets.

Procedure



Figure 1. Glass capillary (A, B) and mannitol droplets on coverslip (C, D). A. A glass capillary drawn by hand. Tip region was boxed. B. Tip opening of the glass capillary in panel A. C and D. A photo (C) and an image (D) of mannitol droplets inside the mineral oil over the siliconized coverslip. Bars = 1 cm in A and C, 200 μm in B.

  1. Sperm cell isolation and microscopic observation of sperm nuclear membrane
    1. Overlay the siliconized coverslip with 0.3 ml mineral oil and make several 1-2 μl mannitol droplets (370 mosmol/kg H2O) inside the mineral oil on the coverslip (Figure 1C) using a glass capillary connected to a handling injector. Take care that the droplets have no access to the air (Figure 1D).
    2. Dissect an unopened flower from the transgenic rice plants expressing SUN2-GFP to obtain anthers under a dissecting microscope (Figure 2A and B).
    3. Transfer anthers into a 3.5 cm plastic dishe filled with 3 ml of mannitol solution (370 mosmol/kg H2O). Break anthers in mannitol solution with forceps to free pollen grains (Figure 2C).
    4. Pollen grains burst due to the osmotic shock and release sperm cells. Identify the sperm cells under an inverted microscope (Figure 3A). By using a glass capillary connected to a handling injector, collect sperm cells and transfer into mannitol droplets prepared in step A1.
    5. SUN2-GFP localizing in nuclear membrane is visualized with an inverted fluorescence microscope with 460-490 nm excitation and 510-550 nm emission wavelengths (Figure 3B and C).


      Figure 2. Preparation of pollen grains from rice flower. A. A mature flower whose lemma and palea were opened. B. A pistil connected with six stamens isolated from the flower in panel A. C. A broken anther and released pollen grains in mannitol solution. Arrows in panels B and C and arrowheads in panels C indicate anthers and pollen grains, respectively. Bars = 5 mm in A and B, 500 µm in C.


      Figure 3. Rice gametes expressing the SUN2-GFP or histone H2B-GFP fusion protein. A. A pollen grain expressing SUN2-GFP released its content in mannitol solution. B and C. Two sperm cells enclosed within the square in panel A were visualized under fluorescence microscopy. Panel C contains the merged bright-field and fluorescent images. D and E. An egg cell isolated from transgenic rice expressing histone H2B-RFP was visualized under bright-field (D) and fluorescence (E) microscopy. Arrow indicates a pollen grain releasing its content. Arrowheads in panels C and E indicate sperm nucleus and egg nucleus, respectively. Bars = 20 μm

  2. In vitro fertilization with rice gametes and LSM observation of gamete nuclear membrane and nucleus in the fused gametes/zygotes
    1. Overlay the glass base dish with 0.2 ml mineral oil, and make a 1-2 μl mannitol droplet (450 mosmol/kg H2O) using a glass capillary connected with handling injector.
    2. Isolate egg cells from transgenic rice plants expressing H2B-RFP according to the previous protocol (Okamoto, 2011). By using a glass capillary connected to a handling injector, transfer an egg cell in a mannitol droplet on a coverslip prepared in step A1 (Figure 3D and E).
    3. Collect sperm cells expressing SUN2-GFP as described in step A, and transfer a sperm cell into a mannitol droplet with an egg cell from step B2.
    4. Electro-fuse the sperm cell and the egg cell in the droplet according the previous protocol (Okamoto, 2011). Briefly, an egg cell was fixed at one electrode under an alternating current field, and a sperm cell was aligned with the egg cell. Cell fusion is induced by applying a single negative direct current pulse, resulting in production of a fused gamete (zygote).
    5. Transfer the resultant zygote to a mannitol droplet on the glass base dish prepared in step B1. Observe the zygote under the LMS 710 CLS microscope using an oil immersion objective lens with 488-nm excitation and 505-530-nm emission wavelengths for SUN2-GFP and with 543-nm excitation and >560-nm emission wavelengths for H2B-RFP.
    6. Obtaining Z-stack serial images of sperm nuclei by LSM observations are described below.
      Image size: 1,024 pixel (X-axis) X 1,024 pixel (Y-axis), Digital zoom: 3.8 fold, Pinhole: 90 μm, Laser power (488-nm): 0.3%, Laser power (543-nm): 0.3%, Number of serial image (Z-axis): 12-16, Z-stack thickness: 0.6 μm


      Figure 4. Three-dimensional reconstruction and volume measurement of sperm nucleus within a zygote. The zygote produced by electro-fusion of a sperm cell expressing SUN2-GFP and an egg cell expressing H2B-RFP was observed under a CLS microscope. A. The 3D image automatically constructed by the IMARIS software using Z-stack images from CLS microscope observation. B. Magnified image of panel A. C. Outline of sperm nucleus, represented in pink colored dotted line, was marked by spotting points on the SUN2-GFP labeled nuclear membrane for each Z-stack images. D. Merged outlines of a sperm nucleus from six Z-stack images. E. Merged outlines for a sperm nucleus composed of 12 Z-stack images. F. The 3D image constructed from the merged outlines of a sperm nucleus. The number in the panel represents the volume of sperm nucleus. G. The 3D image of sperm and egg nuclei. Bars = 10 μm in A, 5 μm in B-G

  3. Three-dimensional reconstruction and volume measurements of sperm nucleus during karyogamy
    1. Open Z-stack serial images of sperm nuclear membrane using IMARIS software. A 3D image is automatically generated (Figure 4A).
    2. Adjust the size of the 3D image for image manipulation (software operation: Edit-Crop 3D) (Figure 4B).
    3. Filter out noise signal from the 3D image created in step C2 (software operation: Image processing-Image smoothing).
    4. In the manual 3D construction mode (software operation: Surpass-Surface-Manual), generate an outline of sperm nucleus by spotting points on the SUN2-GFP labeled nuclear membrane for each Z-stack image (software operation: Draw-Contour-Mode- Click) (Figure 4C-E).
    5. Reconstruct the 3D image using the manually outlined sperm nucleus from step C4 (software operation: Create surface) (Figure 4E-F), and measure the volume of sperm cell nucleus (software operation: Imaris Maesurement Pro-Volume) (Figure 4F-G).

Notes

  1. Rice plants are grown in environmental chamber at 26 °C in a 13/11 h light/dark cycle with a photosynthetic photon flux density of 150-300 μmol photons/m2/s.
  2. Glass of the glass base dishes should be non-coated, as using coated glass base will result in attachment of the cells to the surface of the glass.
  3. Coverslips should be also non-coated, as using coated coverslips will result in attachment of the cells to the surface of the coverslip.
  4. Tip openings were drawn by hand to 150-250 μm.

Acknowledgments

This work was supported, in part, by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Nos. 21112007 and 26113715 to T.O.) and from the Japan Society for the Promotion of Science (No. 25650083 to T.O.). We thank Ms. Hiroki Maeda and Ms. Tomoko Mochizuki (Tokyo Metropolitan University) for technical assistance, and Dr. Min Yvonne Kim (University of California, Berkeley) for critical reading of the protocol.

References

  1. Ohnishi, Y., Hoshino, R. and Okamoto, T. (2014). Dynamics of male and female chromatin during karyogamy in rice zygotes. Plant Physiol 165(4): 1533-1543.
  2. Okamoto T. (2011). In vitro fertilization with isolated rice gametes: production of zygotes and zygote and embryo culture. Methods Mol Biol 710: 17-27.
  3. Uchiumi, T., Komatsu, S., Koshiba, T. and Okamoto, T. (2006). Isolation of gametes and central cells from Oryza sativa L. Sex Plant Reprod 19(1): 37-45.
  4. Uchiumi, T., Uemura, I. and Okamoto, T. (2007). Establishment of an in vitro fertilization system in rice (Oryza sativa L.). Planta 226(3): 581-589.

简介

核型,精子核朝向卵核和它们随后的核融合的迁移是引起被子植物中早期胚胎形成的合子形成的重要生物事件。然而,男性核如何接近和融合与女性核仍然不清楚。最近,核染色体事件中核体积的延时测量显示精子核在与卵核接触期间扩大,通过卵染色质可能的一向迁移到精子核中(Ohnishi等人,2014年, )。在这里,我们描述了用于显微观察,三维重建和体积测量的水稻合子/融合配子中的精子细胞核的方案,其由体外受精系统产生(Uchiumi等人al。,2006; Uchiumi等人,2007)。本协议将适用于监测细胞分裂,分化,去分化和极性形成以及核型进展期间细胞中的核动力学。

关键字:卵细胞, 核融合, 核聚变, 精子细胞, 受精卵

材料和试剂

  1. 分别靶向核膜或核染色质的表达SUN2-GFP或H2B-RFP融合蛋白的转基因水稻植物(水稻(Oryza sativa)L.cv.Nipponbare)(Ohnishi等人, em>,2014)(见注1)
  2. 甘露醇(Wako Chemicals USA,目录号:133-00845)
  3. 370 mosmol/kg H 2 O(330mM)甘露醇溶液(高压灭菌)
  4. 450 mosmol/kg H 2 O(385mM)甘露醇溶液(高压灭菌)
  5. 矿物油(Sigma-Aldrich,目录号:M8410-100ML)

设备

  1. 倒置荧光显微镜(OLYMPUS,型号:BX-71)
  2. 共聚焦激光扫描(CLS)显微镜(ZEISS,型号:LMS 710)
  3. 未处理的直径为3.5cm的塑料盘(Iwaki,目录号:1000-035)
  4. 直径3.5厘米的玻璃底盘(Iwaki,目录号:3971-035)(见注2)
  5. 盖玻片(24×40mm)(Thermo Fisher Scientific,目录号:125485J)(在边缘处用5%二氯甲基硅烷在1,1,1-三氯乙烷中硅化)(见注3)
  6. 由50μl抽吸管(Drummond Scientific Company,目录号:2-000-050)(图1A-B)(见注4)制成的玻璃毛细管
  7. 手动处理喷油器(ST Science,型号UJB)
  8. 环境室(Koito Industries Ltd.,目录号:K30-7248)

软件

  1. IMARIS软件(位平面)
    注意:一个核心的科学软件模块,提供3D和4D显微数据集的数据管理,可视化,分析,分段和解释所需的功能。

程序



图1.玻璃毛细管(A,B)和盖玻片上的甘露醇液滴(C,D)。 A.手工绘制的玻璃毛细管。 尖端区域被加框。 B.面板A中的玻璃毛细管的尖端开口.C和D.在硅化盖片上的矿物油内部的甘露醇小滴的照片(C)和图像(D)。 棒= A和C中1cm,B中200μm

  1. 精子细胞分离和显微镜观察精子核膜
    1. 覆盖硅化盖玻片与0.3毫升矿物油,使 几个1-2微升的甘露醇液滴(370 mosmol/kg H 2 O) 油在盖玻片上(图1C)使用连接到a的玻璃毛细管 处理注射器。 注意液滴不能接触空气   (图1D)。
    2. 解剖来自转基因水稻的未开花   在解剖下表达SUN2-GFP的植物获得花药 显微镜(图2A和B)
    3. 转移花药到3.5厘米 填充有3ml甘露醇溶液(370 mosmol/kg H 2 O)的塑料容器中。   打破花药在甘露醇溶液用镊子释放花粉粒 (图2C)。
    4. 花粉颗粒由于渗透休克而爆裂 释放精子细胞。 识别倒置下的精子细胞 显微镜(图3A)。 通过使用连接到a的玻璃毛细管 处理注射器,收集精细胞并转移到甘露醇 在步骤A1中制备的液滴
    5. SUN2-GFP定位于核 膜用倒置荧光显微镜观察 460-490nm激发和510-550nm发射波长(图3B和   C)。


      图2.从水稻花中制备花粉颗粒。A 成熟的花,其外a和内襟被打开。 B.雌蕊连接 与六个雄蕊隔离从花在面板A. C.破碎的花药   并释放花粉粒在甘露醇溶液中。 面板B和中的箭头   C和箭头C指示花药和花粉粒, 分别。 棒=在A和B中5mm,在C中500μm

      图3. Rice 表达SUN2-GFP或组蛋白H2B-GFP融合蛋白的配子。 A. A 花粉粒表达SUN2-GFP释放其内容物在甘露醇中 解。 B和C.两个精子细胞封闭在图A中的正方形内   在荧光显微镜下观察。 面板C包含 合并明场和荧光图像。 D和E.一个卵细胞 从表达组蛋白H2B-RFP的转基因稻中分离 在明场(D)和荧光(E)显微镜下。 箭头表示a   花粉粒释放其内含物。 图C和E中的箭头 分别表示精子核和卵核。 条=20μm

  2. 体外受精用配子和LSM观察配子核膜和融合的配子/合子中的核
    1. 覆盖玻璃基盘与0.2毫升矿物油,并使一个1-2微升 甘露醇液滴(450 mosmol/kg H 2 O)使用连接的玻璃毛细管 与操作注射器。
    2. 从转基因水稻分离卵细胞 根据先前方案表达H2B-RFP的植物(Okamoto, 2011)。 通过使用连接到处理注射器的玻璃毛细管, 将制备的盖玻片上的甘露醇小滴中的卵细胞转移 步骤A1(图3D和E)。
    3. 收集精子细胞表达 SUN2-GFP,并将精细胞转移到步骤B. 甘露醇液滴与来自步骤B2的卵细胞接触
    4. 电熔丝 精子细胞和卵细胞的液滴中 协议(Okamoto,2011)。 简言之,将卵细胞固定在一个 电极在交流电场下,和精子细胞 与卵细胞对齐。 通过应用单个细胞诱导细胞融合 负直流脉冲,导致产生融合配子   (合子)。
    5. 将所得合子转移至甘露醇液滴 在步骤B1中制备的玻璃底盘上。 观察下的zygote   LMS 710 CLS显微镜使用带有浸油物镜的 488nm激发和505-530nm发射波长的SUN2-GFP和 对于H2B-RFP具有543nm激发和> 560nm发射波长。
    6. 下面描述通过LSM观察获得精子细胞核的Z-堆叠系列图像 图像尺寸:1,024像素(X轴)X 1,024像素(Y轴),数码变焦: 3.8倍,针孔:90μm,激光功率(488nm):0.3%,激光功率 (543nm):0.3%,串行图像数(Z轴):12-16,Z堆叠 厚度:0.6μm


      图4.三维重建和 受精卵内精子核的体积测量。产生的受精卵  通过电融合表达SUN2-GFP的精细胞和卵细胞 在CLS显微镜下观察表达H2B-RFP。 A. 3D图像 由IMARIS软件使用Z-stack图像自动构建 从CLS显微镜观察。 B.面板A的放大图像 以粉红色的虚线表示的精子细胞核的轮廓是 标记在SUN2-GFP标记的核膜上的点样点 每个Z堆栈图像。 D.从六个精子细胞核合并轮廓 Z堆栈图像。 E.由12个精子核组成的合并轮廓 Z堆栈图像。 F.从a的合并轮廓构建的3D图像  精子细胞核。面板中的数字表示精子的体积 核。 G.精子和卵核的3D图像。棒=10μm中的A,5 μm

  3. 核色体中精子核的三维重建和体积测量
    1. 使用IMARIS软件打开精子核膜的Z-堆叠系列图像。 自动生成3D图像(图4A)。
    2. 调整3D图像的大小以进行图像处理(软件操作:Edit-Crop 3D)(图4B)。
    3. 从在步骤C2(软件操作:图像处理 - 图像平滑)中创建的3D图像中滤除噪声信号。
    4. 在手动3D构造模式(软件操作: Surpass-Surface-Manual),通过生成精子核的轮廓 每个在SUN2-GFP标记的核膜上的点 Z-stack图像(软件操作:绘制轮廓模式点击)(图 4C-E)。
    5. 使用手动轮廓重建3D图像 精子核从步骤C4(软件操作:创建表面)(图 4E-F),并测量精子细胞核的体积(软件操作:   Imaris Maesurement Pro-Volume)(图4F-G)。

笔记

  1. 水稻植物在环境室中在26℃在13/11小时光/暗循环中生长,光合通量密度为150-300μmol光子/m 2/s/s。
  2. 玻璃底盘的玻璃应该是未涂覆的,因为使用涂覆的玻璃底座将导致细胞附着到玻璃的表面。
  3. 盖玻片也应该是未涂覆的,因为使用涂覆的盖玻片将导致细胞附着到盖玻片的表面。
  4. 用手将尖端开口拉至150-250μm

致谢

这项工作得到了日本教育,文化,体育,科学和技术部(第21112007和26113715至TO)部和来自日本促进科学协会 TO的第25650083号)。 我们感谢Hiroki Maeda女士和Mochizok女士(东京都城大学)的技术援助,和Min Yvonne Kim博士(加州大学伯克利分校)的关键阅读的协议。

参考文献

  1. Ohnishi,Y.,Hoshino,R。和Okamoto,T.(2014)。 水稻合子中karyogamy期间的雄性和雌性染色质的动态。植物生理学 165(4):1533-1543。
  2. Okamoto T.(2011)。 体外施用分离的水稻配子:产生受精卵和受精卵, 胚胎培养。 Methods Mol Biol 710:17-27。
  3. Uchiumi,T.,Komatsu,S.,Koshiba,T.and Okamoto,T。(2006)。 从水稻中分离配子和中心细胞 L Sex Plant Reprod 19(1):37-45
  4. Uchiumi,T.,Uemura,I。和Okamoto,T。(2007)。 在水稻中建立体外受精系统( Oryza sativa L.)。 Planta 226(3):581-589。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Ohnishi, Y. and Okamoto, T. (2015). Microscopic Observation, Three-dimensional Reconstruction, and Volume Measurements of Sperm Nuclei. Bio-protocol 5(7): e1437. DOI: 10.21769/BioProtoc.1437.
  2. Ohnishi, Y., Hoshino, R. and Okamoto, T. (2014). Dynamics of male and female chromatin during karyogamy in rice zygotes. Plant Physiol 165(4): 1533-1543.
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