Name: Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization (Video) | JoVE Journal
Uploaded: 2019-08-29T14:00:00.000+00:00
Duration: 5 s
Description: Learn to assess fertilization status in Arabidopsis by tracing sperm nuclear morphology during double fertilization. This method enables rapid data collection for statistical analysis of unknown fertilization regulators.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This work was supported by Japan Society for the Promotion of Science KAKENHI grant (JP17H05832 to T. I.) and by funding from the Strategic Priority Research Promotion Program on Phytochemical Plant Molecular Sciences, Chiba University (Japan).

1. Artificial Pollination
NOTE: Before starting the process, a pair of No. 5 forceps is required.
<ol>
	<li>Grow A. thaliana (Col-0) at 22 &#176;C under a 16-h light/8-h dark cycle in a growth chamber. 
	NOTE: Cut and remove the first developed flower stalk with scissors to promote development of axillary buds. Vigorously growing plants (2-3 weeks after cutting of the first stalk; plant height about 25 cm) are suitable for analysis.</li>
	<li>To emasculate, remove sepal (Figure 1B), petal (Figure 1C), and stamen (Figure 1D) of flower buds at stage 118 (Figure 1A) using No. 5 forceps. Bud with bits of petals seen at the top is best. 
	NOTE: Use a suitable female gamete marker line. In this protocol, we used a wild type plant as the female parent.</li>
	<li>Fifteen to eighteen hours after emasculation, take the stamen of a DMP9KD/HTR10-mRFP flower at stage 138 by pinching the filament with forceps.</li>
	<li>To pollinate, gently pat the stigma of an emasculated pistil several times with a dehiscent anther.</li>
</ol>
2. Preparation of Ovule for Observation
NOTE: The following items are required: a slide glass with double-sided tape attached, No. 5 forceps, a 27 G injection needle, and a dissecting microscope.
<ol>
	<li>7 to 8 h after pollination (HAP), collect the pistil and place it on the double-sided tape, then press gently with forceps to fix the pistil on the tape (Figure 2A,A&#8217;). 
	NOTE: Most ovules in a pistil receive at least one pollen tube 10 HAP9. If both or any one of the sperm cells from the first pollen tube fail to fertilize, a second pollen tube would be attracted by the ovule due to the fertilization recovery system10. To analyze the sperm nuclei morphology from the first pollen tube, it is recommended to complete ovule preparation by 10 HAP at the latest.</li>
	<li>Cut off the upper and lower ends of the ovary using an injection needle under a dissecting microscope (Figure 2B,B&#8217;).</li>
	<li>Slit the ovary wall along both sides of the replum (Figure 2C,C&#8217;) by moving the tip of the injection needle. 
	NOTE: Insert the injection needle shallowly to prevent ovule separation from the septum.</li>
	<li>Evert the ovary wall by using the injection needle (Figure 2D,D&#8217;).</li>
	<li>Pinch the base of the septum to which ovules are connected, and lift it up carefully with forceps (Figure 2E).</li>
	<li>Transfer the ovules into a drop of water on a slide glass, and gently cover with a cover glass for observation under a fluorescence microscope (Figure 2E,E&#8217;).</li>
</ol>
3. Microscopy
NOTE: In this protocol, we used an epifluorescence microscope equipped with a fluorescence filter cube (see Table of Materials), a digital camera, and the accompanying software.
<ol>
	<li>Acquire images of ovules containing sperm nuclei labeled with mRFP using a 20x or 40x objective lens and the equipped digital camera.</li>
	<li>Confirm the number of mRFP-labeled sperm nuclei in an embryo sac. 
	NOTE: Ovules containing two mRFP-labeled sperm nuclei can be included in the population size for statistical analysis.</li>
	<li>Confirm the shape and position of each mRFP-labeled sperm nucleus in an embryo sac. 
	NOTE: Immediately after being released from a pollen tube, a pair of condensed mRFP-labeled sperm nuclei are localized between the egg and the central cell. A decondensed mRFP-labeled sperm nucleus detected at the side of chalazal end indicates central cell fertilization, for instance. By using a suitable female gamete membrane marker line, as shown in Supplementary Figure 1, whether or not the sperm cell is undergoing plasmogamy (after membrane fusion but before karyogamy) can be monitored clearly.</li>
</ol>

<ol><li>Mori, T., Kawai-Toyooka, H., Igawa, T., Nozaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gamete+dialogs+in+green+lineages%5BTitle%5D)+AND+%22Molecular+Plant%22%5BJournal%5D)">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/pubmed?term=((Fertilization+mechanisms+in+flowering+plants%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">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/pubmed?term=((GENERATIVE+CELL+SPECIFIC+1+is+essential+for+angiosperm+fertilization%5BTitle%5D)+AND+%22Nature+Cell+Biology%22%5BJournal%5D)">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/pubmed?term=((Arabidopsis+HAP2+%28GCS1%29+is+a+sperm-specific+gene+required+for+pollen+tube+guidance+and+fertilization%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">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/pubmed?term=((Gamete+attachment+requires+GEX2+for+successful+fertilization+in+Arabidopsis%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">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/pubmed?term=((The+male+gamete+membrane+protein+DMP9%2FDAU2+is+required+for+double+fertilization+in+flowering+plants%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">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/pubmed?term=((Distinct+dynamics+of+HISTONE3+variants+between+the+two+fertilization+products+in+plants%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">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/pubmed?term=((Early+flower+development+in+Arabidopsis%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">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/pubmed?term=((Fertilization+recovery+system+is+dependent+on+the+number+of+pollen+grains+for+efficient+reproduction+in+plants%5BTitle%5D)+AND+%22Plant+Signaling+%26+Behavior%22%5BJournal%5D)">Fertilization recovery system is dependent on the number of pollen grains for efficient reproduction in plants.</a> Plant Signaling & Behavior. 8, e23690(2013).</li>
<li>Kasahara, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fertilization+recovery+after+defective+sperm+cell+release+in+Arabidopsis%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">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/pubmed?term=((Live-cell+imaging+reveals+the+dynamics+of+two+sperm+cells+during+double+fertilization+in+Arabidopsis+thaliana%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">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/pubmed?term=((Analysis+of+gamete+membrane+dynamics+during+double+fertilization+of+Arabidopsis%5BTitle%5D)+AND+%22Journal+of+Plant+Research%22%5BJournal%5D)">Analysis of gamete membrane dynamics during double fertilization of Arabidopsis.</a> Journal of Plant Research. 126, 387-394 (2013).</li>
</ol>

The authors have nothing to disclose.

HTR10-mRFP labels paternal chromatin (i.e., visualizes sperm cell nuclei), and the dynamics in double fertilization have been reported7. Immediately after release from a pollen tube, HTR10-mRFP-labeled sperm nuclei are still condensed. However, each of the sperm nuclei is decondensed upon merging with a fertilized female gamete nucleus at karyogamy three to four hours after gamete membrane fusion7. Unfertilized sperm cells remain condensed, as shown in an embryo sac in whic...

Flowering plants produce seeds through double fertilization, a process that is directly controlled by interactions between proteins localized on gamete plasma membrane1,2. Flowering plant male gametes, a pair of sperm cells, develop in pollen. A pollen tube that grows after pollination delivers a pair of sperm cells to female gametes, an egg cell and a central cell, which develop in an embryo sac. After the male and female gametes meet, proteins on the gamete surface promote recognition, attachment, and fusion to complete double fertilization. In previous studies, the male gamete membrane proteins GENERATIVE CELL SPECIFIC 1 (GCS1)/HAPLESS2 (HAP2)3,4 and GAMETE EXPRESSED 2 (GEX2)5 were identified as fertilization regulators involved in gamete fusion and attachment, respectively. We recently identified a male gamete-specific membrane protein, DUF679 DOMAIN MEMBRANE PROTEIN 9 (DMP9), as a fertilization regulator involved in gamete interaction. We found that a decrease of DMP9 expression results in significant inhibition of egg cell fertilization during double fertilization in A. thaliana6.
As double fertilization occurs in an embryo sac, which is embedded in an ovule that is further wrapped with ovary tissue, it is difficult to observe and analyze the states of double fertilization processes. For this reason, there are still many unclear points that hinder a complete understanding of the whole mechanism of double fertilization control. The establishment of observation techniques to trace the behavior of gametes during double fertilization under in vivo conditions is indispensable for the functional analysis of potential candidates for fertilization regulators. Recent studies have yielded marker lines where gamete subcellular structures are labeled with fluorescent proteins. In this article, we demonstrate a simple and quick protocol for observing double fertilization that has occurred in an embryo sac derived from artificially pollinated pistils. Using sperm cell nucleus marker line HTR10-mRFP7, the fertilization state of each female gamete can be discriminated on the basis of sperm nuclear signal morphology. Our protocol focusing on such a morphological change of the sperm nuclei at fertilization can efficiently obtain a sufficient amount of data for statistical proof. A DMP9-knockdown line with HTR10-mRFP background (DMP9KD/HTR10-mRFP) was used as male plants to show a single fertilization pattern. The protocol is also suitable for the functional analysis of other fertilization regulators.

<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>&lt;em&gt;DMP9&lt;sup&gt;KD/HTR10-mRFP&lt;/sup&gt;&lt;/em&gt;</td><td></td><td></td><td>&lt;em&gt;Arabidopsis thaliana&lt;/em&gt;, &lt;em&gt;HTR10-mRFP&lt;/em&gt; background&lt;br/&gt; Takahashi et al. (2018)&lt;sup&gt;6&lt;/sup&gt;</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>&lt;em&gt;HTR10-mRFP&lt;/em&gt;</td><td></td><td></td><td>&lt;em&gt;Arabidopsis thaliana&lt;/em&gt;, ecotype Columbia-0 (Col-0) background&lt;br/&gt; Ingouff et al. (2007)&lt;sup&gt;7&lt;/sup&gt;</td></tr><tr><td>Injection needle</td><td>Terumo</td><td>NN-2719S</td><td>27 G</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 (NA 0.75), dry</td></tr></tbody></table>

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

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

Flowering plants have a unique sexual reproduction system called &#8216;double fertilization&#39;,&#160;in which each of the sperm cells precisely fuses with an egg cell or a central cell. Thus, two independent fertilization events take place almost simultaneously. The fertilized egg cell and central cell develop into zygote and endosperm, respectively. Therefore, precise control of double fertilization is essential for the ensuing seed development. Double fertilization occurs in the female gametophyte (embryo sac), which is deeply hidden and covered with thick ovule and ovary tissues. This pistil tissue construction makes observation and analysis of double fertilization quite difficult and has created the present situation in which many questions regarding the mechanism of double fertilization remain unanswered. For the functional evaluation of a potential candidate for fertilization regulator, phenotypic analysis of fertilization is important. To judge the completion of fertilization in Arabidopsis thaliana, the shapes of fluorescence signals labeling sperm nuclei are used as indicators. A sperm cell that fails to fertilize is indicated by a condensed fluorescence signal outside of the female gametes, whereas a sperm cell that successfully fertilizes is indicated by a decondensed signal due to karyogamy with the female gametes&#8217; nucleus. The method described here provides a tool to determine successful or failed fertilization under in vivo conditions.

Ovules from a pistil pollinated with DMP9KD/HTR10-mRFP were collected at 7-8 HAP and observed.
Most ovules contained two decondensed mRFP-labeled sperm nuclei at the egg cell (micropylar side) and central cell (charazal end side) nucleus positions, respectively (Figure 3A), indicating successful double fertilization. In addition, ovules containing a decondensed mRFP-labeled sperm...

We demonstrate a method to determine successful or failed fertilization on the basis of sperm nuclear morphology in Arabidopsis double fertilization using an epifluorescence microscope.

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

evaluation-fertilization-state-tracing-sperm-nuclear-morphology

Research

JoVE Journal

Developmental Biology

Imprinting is a phenomenon in plant and mammal reproduction. DNA methylation plays an important role in mechanisms of imprinting. Isolating endosperm and determining methylation status of imprinted genes in Arabidopsis can be difficult. In this protocol, we describe how to isolate endosperm and determine methylation by bisulfite sequencing.

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

This protocol outlines a method for observing embryogenesis in Arabidopsis via ovule clearance followed by the inspection of embryo pattern formation under a microscope.

A Method for Characterizing Embryogenesis in Arabidopsis

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In this protocol, we describe techniques for the proper dissection of Arabidopsis flowers and siliques, some basic clearing techniques, and selected staining procedures for whole-mount observations of reproductive structures.

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Here, we describe an improvement of the semi-in vitro (SIV) method for observing pollen tube guidance and reception in Arabidopsis thaliana, which increases the receptivity of ovules. The high-throughput SIV cum septum method may be coupled with gametophyte marker lines and genetically encoded biosensors to monitor the dynamic process of fertilization.

Live Imaging of Arabidopsis Pollen Tube Reception and Double Fertilization Using the Semi-In Vitro Cum Septum Method

An improved method to measure pollen hydration profiles in Arabidopsis thaliana is described here. The new method offers higher resolution, is noninvasive, and is highly reproducible. The protocol represents a new tool for a finer dissection of the processes that regulate the early stages of pollination.

A High-Resolution, Single-Grain, In Vivo Pollen Hydration Bioassay for Arabidopsis thaliana

This protocol describes the preparation of intact samples of the endosperm cell layer in Arabidopsis thaliana seeds. The method requires only common laboratory equipment, such as an injection needle and precision forceps, and enables high-resolution fluorescent live-cell imaging of endosperm cells in both developing and mature seeds.

Preparation of Intact Tissue for Microscopic Analysis of the Endosperm Cell Layer in Developing and Mature Arabidopsis Seeds

Chromosome movement during meiotic prophase is a poorly understood phenomenon that is fundamental to spore formation. This protocol describes a method to acquire, analyze, and quantify centromere movements during meiotic prophase in Arabidopsis thaliana male meiocytes.

Live Imaging Characterization of Centromere Movements During Male Meiotic Prophase in Arabidopsis thaliana

Learn to assess fertilization status in Arabidopsis by tracing sperm nuclear morphology during double fertilization. This method enables rapid data collection for statistical analysis of unknown fertilization regulators.

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

Explore More Videos