Name: evaluation of fertilization state by tracing sperm nuclear morphology in arabidopsis double fertilization
Uploaded: 2019-08-29T14:00:00
Description: Watch this Scientific Journal Video about Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization at JoVE.com

这项工作得到了日本科学促进协会KAKENHI赠款（JP17H05832至T.I.）的支持，以及日本千叶大学植物化学植物分子科学战略优先研究促进方案的资助。

1. 人工授粉
注：在开始该过程之前，需要一对 5 号钳子。
<ol><li>在生长室的16-h光/8-h暗循环下，在22°C下生长A.thaliana（Col-0）。 注：用剪刀切割和去除第一个开发的花茎，以促进开枝芽的发展。大力种植植物（切割第一茎后2-3周;植物高度约25厘米）适合分析。</li>
	<li>要削弱花蕾（图1B）、花瓣（图1C）...

<ol>
	<li>Mori, T., Kawai-Toyooka, H., Igawa, T., Nozaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gamete+dialogs+in+green+lineages.">Gamete dialogs in green lineages.</a> Molecular Plant. 8, 1442-1454 (2015).</li><li>Dresselhaus, T., Sprunck, S., Wessel, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertilization+mechanisms+in+flowering+plants.">Fertilization mechanisms in flowering plants.</a> Current Biology. 26, R125-R139 (2016).</li><li>Mori, T., Kuroiwa, H., Higashiyama, T., Kuroiwa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GENERATIVE+CELL+SPECIFIC+1+is+essential+for+angiosperm+fertilization.">GENERATIVE CELL SPECIFIC 1 is essential for angiosperm fertilization.</a> Nature Cell Biology. 1, 64-71 (2006).</li><li>von Besser, K., Frank, A. C., Johnson, M. A., Preuss, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+HAP2+(GCS1)+is+a+sperm-specific+gene+required+for+pollen+tube+guidance+and+fertilization.">Arabidopsis HAP2 (GCS1) is a sperm-specific gene required for pollen tube guidance and fertilization.</a> Development. 133, 4761-4769 (2006).</li><li>Mori, T., Igawa, T., Tamiya, G., Miyagishima, S. Y., Berger, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gamete+attachment+requires+GEX2+for+successful+fertilization+in+Arabidopsis.">Gamete attachment requires GEX2 for successful fertilization in Arabidopsis.</a> Current Biology. 24, 170-175 (2014).</li><li>Takahashi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+male+gamete+membrane+protein+DMP9/DAU2+is+required+for+double+fertilization+in+flowering+plants.">The male gamete membrane protein DMP9/DAU2 is required for double fertilization in flowering plants.</a> Development. 145, dev170076 (2018).</li><li>Ingouff, M., Hamamura, Y., Gourgues, M., Higashiyama, T., Berger, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+dynamics+of+HISTONE3+variants+between+the+two+fertilization+products+in+plants.">Distinct dynamics of HISTONE3 variants between the two fertilization products in plants.</a> Current Biology. 17, 1032-1037 (2007).</li><li>Smyth, D. R., Bowman, J. L., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+flower+development+in+Arabidopsis.">Early flower development in Arabidopsis.</a> Plant Cell. 2, 755-767 (1990).</li><li>Kasahara, R. D., Maruyama, D., Higashiyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertilization+recovery+system+is+dependent+on+the+number+of+pollen+grains+for+efficient+reproduction+in+plants.">Fertilization recovery system is dependent on the number of pollen grains for efficient reproduction in plants.</a> Plant Signaling &amp; Behavior. 8, e23690 (2013).</li><li>Kasahara, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertilization+recovery+after+defective+sperm+cell+release+in+Arabidopsis.">Fertilization recovery after defective sperm cell release in Arabidopsis.</a> Current Biology. 22, 1084-1089 (2012).</li><li>Hamamura, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+reveals+the+dynamics+of+two+sperm+cells+during+double+fertilization+in+Arabidopsis+thaliana.">Live-cell imaging reveals the dynamics of two sperm cells during double fertilization in Arabidopsis thaliana.</a> Current Biology. 21, 497-502 (2011).</li><li>Igawa, T., Yanagawa, Y., Miyagishima, S., Mori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+gamete+membrane+dynamics+during+double+fertilization+of+Arabidopsis.">Analysis of gamete membrane dynamics during double fertilization of Arabidopsis.</a> Journal of Plant Research. 126, 387-394 (2013).</li></ol>

HTR10-mRFP标签父色素（即，可视化精子细胞核），和动态在双受精已报告7。从花粉管中释放后，HTR10-mRFP标记的精子核仍然凝结。然而，每个精子核在与受精的雌性体细胞核合并后，在Gamete膜融合7后三到四个小时被解压缩。未受精的精子细胞保持浓缩，如在胚胎囊中所示，在胚胎囊中gcs1精子细胞在没有受精的情况下被吸收（图3D）。...

开花植物通过双重受精产生种子，这个过程由在Gamete血浆膜1、2上局部的蛋白质之间的相互作用直接控制。开花植物雄性配子，一对精子细胞，在花粉中发育。授粉后生长的花粉管将一对精子细胞输送到雌性配子、卵细胞和中心细胞，这些细胞在胚胎囊中发育。雄性配子和雌性配子相遇后，配子表面的蛋白质促进识别、附着和融合，以完成双重受精。在以前的研究中，雄性Gamete膜蛋白：2（GCS1）/HAPLESS2（HAP2）3、4和GAMETE表达2（GEX2）5被确定为参与Gamete融合的受精调节剂，附件。我们最近鉴定出一种雄性gamete特异性膜蛋白，DUF679 DOMAIN，第9蛋白（DMP9），作为参与Gamete相互作用的受精调节剂。我们发现，DMP9表达的减少导致在A.thaliana6的双重受精期间对卵细胞受精的显著抑制。
由于双受精发生在胚胎囊中，胚胎囊嵌入在卵巢组织进一步包裹的卵子中，因此很难观察和分析双重受精过程状态。因此，仍有许多不明确的点，阻碍对双施肥控制的整体机制的全面了解。建立观察技术，以跟踪在体内条件下的双重受精过程中配子的行为，对于对施肥调节器的潜在候选物进行功能分析是必不可少的。最近的研究已经产生了标记线，其中Gamete亚细胞结构被标记与荧光蛋白。在本文中，我们演示了一种简单而快速的协议，用于观察从人工授粉的皮斯蒂尔衍生的胚胎囊中发生的双重受精。使用精子细胞核标记线HTR10-mRFP7，每个雌性胶合物的受精状态可以基于精子核信号形态进行区分。我们的方案侧重于精子核在受精时的变化，可以有效地获得足够的数据进行统计证明。具有HTR10-mRFP背景（DMP9 KD/HTR10-mRFP）的DMP9-敲落线用作雄性植物，以显示单一施肥模式。该协议也适用于其他施肥调节器的功能分析。

<table>
	<tbody>
		<tr>
			<td>BX51</td>
			<td>Olympus</td>
			<td></td>
			<td>Epifluorescence microscope</td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Matsunami glass</td>
			<td>C018181</td>
			<td></td>
		</tr>
		<tr>
			<td>DMP9KD/HTR10-mRFP</td>
			<td></td>
			<td></td>
			<td>Arabidopsis thaliana, HTR10-mRFP background 
			Takahashi et al. (2018)6</td>
		</tr>
		<tr>
			<td>Double-sided tape</td>
			<td>Nichiban</td>
			<td>NW-15S</td>
			<td>15 mm width</td>
		</tr>
		<tr>
			<td>DP72</td>
			<td>Olympus</td>
			<td></td>
			<td>Degital camera</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Vigor</td>
			<td></td>
			<td>Any No. 5 forceps are available</td>
		</tr>
		<tr>
			<td>Growth chamber</td>
			<td>Nihonika</td>
			<td>LPH-411PFQDT-SP</td>
			<td></td>
		</tr>
		<tr>
			<td>HTR10-mRFP</td>
			<td></td>
			<td></td>
			<td>Arabidopsis thaliana, ecotype Columbia-0 (Col-0) background 
			Ingouff et al. (2007)7</td>
		</tr>
		<tr>
			<td>Injection needle</td>
			<td>Terumo</td>
			<td>NN-2719S</td>
			<td>27 gauge</td>
		</tr>
		<tr>
			<td>Slide glass</td>
			<td>Matsunami glass</td>
			<td>S9443</td>
			<td></td>
		</tr>
		<tr>
			<td>SZX9</td>
			<td>Olympus</td>
			<td></td>
			<td>Dissecting microscope</td>
		</tr>
		<tr>
			<td>U-MRFPHQ</td>
			<td>Olympus</td>
			<td></td>
			<td>Fluorescence Filter Cube (Excitation: BP535-555, Emission: BA570-625, Dichromatic mirror:DM565)</td>
		</tr>
		<tr>
			<td>UPlanFL N 40x</td>
			<td>Olympus</td>
			<td></td>
			<td>Objective lens (NA 1.3), oil-immersion</td>
		</tr>
		<tr>
			<td>UPlanSApo 20x</td>
			<td>Olympus</td>
			<td></td>
			<td>Objective lens (NA0.75), dry</td>
		</tr>
	</tbody>
</table>

evaluation of fertilization state by tracing sperm nuclear morphology in arabidopsis double fertilization

开花植物有一个独特的性生殖系统，称为"双受精"，其中每个精子细胞精确与卵细胞或中心细胞融合。因此，两个独立的受精事件几乎同时发生。受精卵细胞和中枢细胞分别发育成酶和内膜。因此，精确控制双施肥对后续种子发育至关重要。双重受精发生在雌性野味（胚胎囊），这是深隐藏，覆盖着厚厚的卵巢和卵巢组织。这种双倍施肥的构造使得对双施肥的观察和分析相当困难，造成了目前双施肥机制问题仍未解决的局面。对于施肥调节器潜在候选者的功能评估，施肥的体称分析非常重要。为了判断在阿拉伯拟南芥中受精的完成情况，用标记精子核的荧光信号的形状作为指标。未能受精的精子细胞由雌性配子外的凝聚荧光信号指示，而成功受精的精子细胞则通过与雌性配子核的结核核的减压信号表示。此处描述的方法提供了一个工具，用于确定在体内条件下成功或失败的受精。

用DMP9 KD/HTR10-mRFP授粉的皮斯蒂尔的卵泡在7-8HAP上被收集并观察。 
大多数卵泡在卵细胞（微皮侧）和中细胞（夏拉扎尔端侧）的细胞核位置分别含有两个解压缩的mRFP标记精子核（图3A），表明成功的双重受精。此外，在中心细胞核处观察到含有解压缩mRFP标记精子核的卵子，以及卵细胞外的?...

Watch this Scientific Journal Video about Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization at JoVE.com

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

我们演示了一种利用荧光显微镜在阿拉伯拟南芥中精子核形态测定成功或失败受精的方法。

通过追踪阿拉伯植物双重受精中的精子核形态对受精国的评价

Flowering plants have a unique sexual reproduction system called &#8216;double fertilization&#39;,&#160;in which each of the sperm cells precisely fuses ...

该协议提供了一种评估某一因素参与阿拉比多普西斯双重受精的方法。受精状态可从精子核的形态中一目了然地判断，因此可以快速获取数据进行统计分析。该方法是特异性评估在阿拉伯表型受精表型。仍然有未知的施肥调节器，即使在阿拉伯。该方法对于未来未知受精因子的功能分析是有用的。掌握快速、完整处理奥武尔的技能对于本分析的成功非常重要。首先，在16小时的灯光下和生长室的8小时黑暗周期下，在22摄氏度下生长阿拉伯沙利亚拉。用剪刀切割和去除第一个开发的花茎，以促进轴芽的发展。切断第一根茎后两到三周，测量植物高度。高度超过25厘米的植物被认为是大力生长的植物，适合分析。要消瘦，在阶段 11 中寻找花蕾，顶部有花瓣。使用数字五钳去除花蕾的花瓣、花瓣和花蕾。消瘦后 15 至 18 小时，通过用钳子捏紧灯丝，在第 13 阶段以 HTR10-mRFP 花背景的 DMP9-敲击线进行固定。要授粉， 轻轻地拍拍一个消瘦的小便的耻辱几次与脱喜的安瑟。授粉后七到八小时，收集皮蒂尔，并放在双面胶带上。然后，用钳子轻轻按压，以固定胶带上的皮斯蒂尔。在解剖显微镜下，使用注射针切断卵巢的上端和下端。移动注射针的尖端，沿凹槽的两侧切开卵巢壁。小心切割，以便不分离真菌和传输道。永远的卵巢墙。捏紧与奥武尔连接到的隔膜的底座，然后用钳子小心地将其提起。将奥弗将转移到滑动玻璃上的一滴水中，并在荧光显微镜下用盖玻璃轻轻盖住，进行观察。在配备荧光滤波立方体的荧光显微镜上，将客观透镜调整为 20 倍或 40 倍。获取含有标有mRFP的精子核的奥武尔的图像。确认胚胎囊中标有 mRFP 标记的精子核的数量。确认胚胎囊中每个 mRFP 标记的精子核的形状和位置。本研究中，受精表型由卵巢精子核信号形态判断。大多数卵子在卵细胞微缩侧和中央细胞字符端侧含有两个脱膜 mRFP 标记的精子核。这表示成功的双重受精。此外，还观察到在中央细胞核中含有脱膜mRFP标记的精子核，以及细胞外一个浓缩的mRFP标记的精子核，表明单一受精。在双受精失败的情况下，在卵细胞和中心细胞的边界处观察到两个浓缩mRFP标记的精子核。在使用具有HTR10-mRFP花粉背景的DMP9-敲击线进行分析时，很少观察到含有单个浓缩mRFP标记精子核或三个或更多mRFP标记精子核的卵子。结合女性标记线的使用，可以分析更准确的受精状态。例如，可以判断游戏融合后和卡约加米之前受精状态。是的，受精过程分为几个步骤。每个施肥调节器所涉及的问题，其中的步骤可以通过这种改进的方法回答。它将提供对植物性繁殖的更深入的了解。

Research

JoVE Journal

Developmental Biology

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

操作和<em&gt;在体外</em中&gt;成熟<em&gt;非洲爪蟾</em&gt;卵母细胞，其次是胞浆内单精子注射，研究胚胎发育

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

科研

发育生物学

Live Confocal Imaging of Developing Arabidopsis Flowers

住发展拟南芥花的共焦成像

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

Bottlenose Dolphin (Tursiops truncatus) Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

Bottlenose Dolphin Tursiops truncatus Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

宽吻海豚 (海豚截形) 精子： 收集、 冷冻保存和外源体外受精

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to ...

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

马卵母细胞染色体分离，组蛋白乙酰化和主轴形态分析

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted ...

A Method for Characterizing Embryogenesis in Arabidopsis

A Method for Characterizing Embryogenesis in Arabidopsis

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, ...

Live Cell Imaging of Chromosome Segregation During Mitosis

有丝分裂过程中染色体分离的活体细胞成像

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Tracing Gene Expression Through Detection of &#946;-galactosidase Activity in Whole Mouse Embryos

Tracing Gene Expression Through Detection of ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-galactosidase Activity in Whole Mouse Embryos

通过检测全小鼠胚胎β乳糖酶活性追踪基因表达

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. ...

Fluorimetric Techniques for the Assessment of Sperm Membranes

精子膜评价的荧光技术

Standard spermiograms describing sperm quality are mostly based on the physiological and visual parameters, such as ejaculate volume and concentration, ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

全斑马鱼胚胎的延长时移共聚焦显微镜的分层安装方法

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues

使用纸屑小鼠研究矿化组织的克隆遗传追踪

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its ...