Name: evaluation of fertilization state by tracing sperm nuclear morphology in arabidopsis double fertilization
Uploaded: 2019-08-29T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization at JoVE.com

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/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>

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 which gcs1 sperm cells are arrested without fertilization (Figure 3D). Usually, when the fertile HTR10-mRFP is used as the pollen parent, 57.9% &#177; 17.8% (mean &#177; s.d.; n = 16 pistils) of ovules in a pistil at 7-8 HAP contain two mRFP-labeled sperm nuclei signals, and almost all of the signals (97.2%) are decondensed, reflecting successful double fertilization (a similar signal pattern is shown in Figure 3A). In the case of DMP9KD/HTR10-mRFP, ovules containing two mRFP-labeled sperm nuclei were observed at a similar frequency to that of HTR10-mRFP, but 17.6% of them showed single fertilization6. We adopted the HTR10-mRFP dynamics for observation of a large number of ovules under in vivo conditions using an epifluorescence microscope. The protocol is simple and quick, which enables the collection of sufficient data for statistical proof. Using this protocol as the first instance, the significance of single fertilization of the central cell by DMP9KD/HTR10-mRFP sperm cells was proved6.
Based on the morphological differences of HTR10-mRFP-labeled sperm nuclei derived from one pollen tube, the fertilization patterns are classified into three phenotypes: (1) successful double fertilization, which is characterized by two decondensed HTR10-mRFP-labeled sperm nuclei (Figure 3A); (2) single fertilization, which features one condensed HTR10-mRFP-labeled sperm nucleus and one decondensed HTR10-mRFP-labeled sperm nucleus (Figure 3B); and (3) failed double fertilization, which has two condensed HTR10-mRFP-labeled sperm nuclei (Figure 3C).
Other potential causes of phenotypes (2) and (3) should be considered. It has been reported that sperm cells respectively begin plasmogamy with an egg cell or central cell several minutes after being released into an embryo sac under semi-in vitro culture conditions11. In addition, a time lag of a few minutes exists between the first and second fertilizations, although there is no preference for the order of fertilization of the egg cell and the central cell11. Therefore, a pair of condensed and decondensed sperm nuclei in phenotype (2) might indicate a time lag between the fertilizations instead of single fertilization. In order to confirm the occurrence of single fertilization at a significant frequency in phenotype (2), a number of samples sufficient for statistical analysis should be examined. Phenotype (3), which has two condensed sperm nuclei (Figure 3C), could reflect a period of immobility of sperm cells prior to plasma membrane fusion12 or the period between plasmogamy and karyogamy. Therefore, two condensed sperm nuclei at 7-8 HAP do not fully reflect &#39;failure&#39; of double fertilization, and it is necessary to observe the ovules at a later time after pollination to assess the success or failure of double fertilization. In this regard, ovule observation at 16-18 HAP is recommended, because most of the ovules would have completed double fertilization with sperm cells delivered by the first pollen tube, and HTR10-mRFP signals of unfertilized sperm nuclei would remain until the next day of pollination, as shown in Figure 3D.
A pattern of single condensed HTR10-mRFP-labeled sperm nucleus (Figure 4A) was rarely detected, which was likely due to the quick disappearance of another paternal HTR10-mRFP signal in the fertilized female gamete as a result of single fertilization. In this analysis, three or more HTR10-mRFP-labeled sperm nuclei were also detected as a rare case (Figure 4B), due to second pollen tube acceptance by the fertilization recovery system10. In this protocol, these cases were excluded from the data, because tracing the behaviors of two sperm cells derived from one pollen tube is critical for accurate assessment.
Since the polarity for the positions of the female gametes in an embryo sac is well regulated (i.e., the egg cell and the central cell differentiate at the micropylar and the chalazal side, respectively), which female gamete is fertilizing can be judged by the relative positions of the mRFP-labeled sperm nuclei. However, there is a limitation in distinguishing between unfertilized and plasmogamy states, because both states are indicated by the condensed sperm nuclei signal. To evaluate precisely whether plasmogamy after gamete membrane fusion has occurred or not when the sperm nuclei are condensed, it is required to use female gamete membrane marker lines, as reported by Takahashi et al. (2018)6. For example, when a pFWA::GFP-PIP plant12 in which the central cell plasma membrane was visualized was used as a female parent, it is clear that a sperm cell fused with the central cell was in plasmogamy (Supplementary Figure 1).
In summary, our protocol described here can be used for statistical assessment of success or failure of fertilization in each female gamete. Although the employment of the female plasma membrane marker is required to judge the plasmogamy, our method is useful for functional analysis of the fertilization regulator.

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

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 nucleus at the central cell nucleus plus a condensed mRFP-labeled sperm nucleus outside the egg cell (Figure 3B) were observed, indicating single fertilization. In the case of failed double fertilization, two condensed mRFP-labeled sperm nuclei were observed at the boundary of the egg cell and the central cell (Figure 3C), as in gcs1 mutant sperm cells (Figure 3D)3,4.
In the analysis using DMP9KD/HTR10-mRFP pollen, ovules containing a single condensed mRFP-labeled sperm nucleus (Figure 4A) or three or more mRFP-labeled sperm nuclei (Figure 4B) were rarely observed.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59916/59916fig1.jpg" /> 
Figure 1: Flower buds suitable for emasculation. (A) A flower bud at stage 11, showing bits of the petals at the top. (B-D) A flower bud in which was removed the sepals (B) and petals (C) and that was then emasculated completely (D). The anther dehiscence has not occurred yet. Scale bar = 1 mm. <a href="https://www.jove.com/files/ftp_upload/59916/59916fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59916/59916fig2.jpg" /> 
Figure 2: Flow of ovule sample preparation. The procedures are shown by illustrations (A-E) and photographs (A&#8217;-E&#8217;). (A, A&#8217;) A pistil is placed on double-sided tape attached to a slide glass, and its upper and lower ends are cut off with an injection needle. (B, B&#8217;) The ovary wall is incised along both sides of the replum. (C, C&#8217;) Both sides of the ovary walls are opened, everted, and pressed onto the tape for fixing. (D, D&#8217;) The septum where ovules are connected in arrays is exposed. (E, E&#8217;) One end of the septum is pinched and lifted up with forceps. The septum with connecting ovules is transferred to a drop of water on a slide glass. <a href="https://www.jove.com/files/ftp_upload/59916/59916fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59916/59916fig3.jpg" /> 
Figure 3: Fertilization phenotypes judged by sperm nuclear signal morphology in ovule. (A) Successful double fertilization indicated by two decondensed mRFP-labeled sperm nuclei (arrowheads). (B) Single fertilization by DMP9KD/HTR10-mRFP sperm cells indicated by one decondensed mRFP-labeled sperm nucleus (arrowhead) and one condensed mRFP-labeled sperm nucleus (arrow). (C) Failed double fertilization by DMP9KD/HTR10-mRFP sperm cells indicated by two condensed mRFP-labeled sperm nuclei (arrows). (D) An example of failed double fertilization by gcs1 HTR10-mRFP sperm cells. A marker line where the egg cell nucleus (EN) is labeled with RFP was used. Two condensed mRFP-labeled sperm nuclei (arrows) are arrested without fertilization at 18 HAP. (E) Schematic illustration of an ovule. EC = egg cell, CC = central cell, SC = synergid cells, ES = embryo sac, MP = micropyle, CHZ = chalazal end. Scale bars =&#160;20 &#956;m. <a href="https://www.jove.com/files/ftp_upload/59916/59916fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59916/59916fig4.jpg" /> 
Figure 4: Other potential fertilization phenotypes. Ovules from a pistil pollinated with DMP9KD/HTR10-mRFP rarely contain a single condensed mRFP-labeled sperm nucleus (A), or three or more condensed mRFP-labeled sperm nuclei (B) (arrows). Scale bars =&#160;20 &#956;m. <a href="https://www.jove.com/files/ftp_upload/59916/59916fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Condensed sperm nuclei at plasmogamy during fertilization, judged by combination with a female plasma membrane marker line. pFWA::GFP-PIP where the central cell plasma membrane is visualized was used as female parent (GFP). GFP-PIP also labels endo membranes12. The ovule contains two condensed mRFP-labeled sperm nuclei (arrows; RFP). The RFP signal at charazal end side (arrow) is shown in the central cell (Merge), indicating that the sperm nucleus is in plasmogamy (after gamete membrane fusion, but before karyogamy). The panel on the right is a magnification of the area surrounded by dashed lines in the merged image. A blank area marked by an asterisk corresponds to the position of the central cell nucleus. The dashed line indicates the outline of the central cell facing the egg cell. Scale bar =&#160;20 &#956;m. <a href="https://www.jove.com/files/ftp_upload/59916/Sup_Figure_1.tif">Please click here to download this file.</a>

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

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.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

8.5K Views.  Chiba University. 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.

Video: 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

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

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

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

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A Method for Characterizing Embryogenesis in Arabidopsis

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

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

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

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

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 most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

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

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, especially, in plant reproductive biology. However, plant morphological, anatomical, and ultrastructural analyses traditionally involve time-consuming embedding and sectioning procedures for bright field, scanning, and electron microscopy. Recent progress in confocal fluorescence microscopy, state-of-the-art 3-D computer-aided microscopic analyses, and the continuous refinement of molecular techniques to be used on minimally processed whole-mount specimens, has led to an increased demand for developing efficient and minimal sample processing techniques. In this protocol, we describe techniques for properly dissecting Arabidopsis flowers and siliques, basic clearing techniques, and some staining procedures for whole-mount observations of reproductive structures.

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

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.

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, ...

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA methyltransferases is viable, thus providing a system to study how loss of DNA methylation in a genome affects growth and development. Imprinting refers to differential expression of maternal and paternal alleles and plays an important role in reproduction development in both mammal and plants. DNA methylation is critical for determining whether the maternal or paternal alleles of an imprinted gene is expressed or silenced. In flowering plants, there is a double fertilization event in reproduction: one sperm cell fertilizes the egg cell to form embryo and a second sperm fuses with the central cell to give rise to endosperm. Endosperm is the tissue where imprinting occurs in plants. MEDEA, a SET domain Polycomb group gene, and FWA, a transcription factor regulating flowering, are the first two genes shown to be imprinted in endosperm and their expression is controlled by DNA methylation and demethylation in plants. In order to determine imprinting status of a gene and methylation pattern in endosperm, we need to be able to isolate endosperm first. Since seed is tiny in Arabidopsis, it remains challenging to isolate Arabidopsis endosperm and examine its methylation. In this video protocol, we report how to conduct a genetic cross, to isolate endosperm tissue from seeds, and to determine the methylation status by bisulfite sequencing.

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

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.

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

Biology

Tracking Calcium in the Early Developmental Phases of C. elegans

In Vivo Visualization of Calcium Transients during Fertilization and Early Development in C. elegans

Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode Caenorhabditis elegans provides several unique advantages for the study of the OET as it is transparent and has an ordered gonad that produces one mature oocyte every ~23 min at 20 &#176;C. We have modified the genetically encoded calcium indicator jGCaMP7s to fluorescently indicate the moment of fertilization within a living organism. We have termed this reporter &#34;CaFE&#34; for Calcium during Fertilization in C. elegans. The CaFE reporter was&#160;engineered into a safe harbor locus in single copy, has no significant impact on physiology or fecundity, and produces a robust signal upon fertilization. Here, a series of protocols is presented for utilizing the CaFE reporter as an in vivo tool for dissecting the OET and embryogenesis. We include methods to synchronize worms, examine the effects of RNAi knockdown, mount worms for imaging, and to visualize calcium in oocytes and embryos. Additionally, we present the generation of additional worm strains to aid in this type of analysis. Demonstrating the utility of the CaFE reporter to visualize the timing of fertilization, we report that double ovulation occurs when ipp-5 is targeted by RNAi and that only the first oocyte undergoes immediate fertilization. Furthermore, the discovery of single-cell calcium transients during early embryogenesis is reported here, demonstrating that the CaFE reporter persists into early development. Importantly, the CaFE reporter in worms is simple enough to use for incorporation into course-based undergraduate research (CURE) laboratory classes. The CaFE reporter, coupled with the ordered gonad and ease of RNAi in worms, facilitates inquiry into the cell-cell dynamics required to regulate internal fertilization and early embryogenesis.

Here, we present protocols and tools for visualizing calcium during fertilization and early embryogenesis using a genetically encoded calcium reporter expressed in the germline of the model nematode C. elegans.

Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode ...

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

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

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

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.

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

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

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

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

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;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.

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

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

In flowering plants, the growth and guidance of the pollen tube (male gametophyte) within the pistil and the reception of the pollen tube by the female gametophyte are essential for double fertilization and subsequent seed development. The interactions between male and female gametophytes during pollen tube reception culminate in pollen tube rupture and the release of two sperm cells to effect double fertilization. As pollen tube growth and double fertilization are deeply hidden within the tissues of the flower, this process is difficult to observe in vivo.
A semi-in vitro (SIV) method for the live-cell imaging of fertilization in the model plant Arabidopsis thaliana has been developed and implemented in several investigations. These studies have helped to elucidate the fundamental features of how the fertilization process occurs in flowering plants and which cellular and molecular changes occur during the interaction of the male and female gametophytes. However, because these live cell imaging experiments involve the excision of individual ovules, they are limited to a low number of observations per imaging session, making this approach tedious and very time-consuming. Among other technical difficulties, a failure of the pollen tubes to fertilize the ovules in vitro is&#160;often reported, which severely confounds such analyses.
Here, a detailed video protocol for the imaging of pollen tube reception and fertilization in an automated and high-throughput manner is provided, allowing for up to 40 observations of pollen tube reception and rupture per imaging session. Coupled with the use of genetically encoded biosensors and marker lines, this method enables the generation of large sample sizes with a reduced time investment. Nuances and critical points of the technique, including flower staging, dissection, medium preparation, and imaging, are clearly detailed in video format to facilitate future research on the dynamics of pollen tube guidance, reception, and double fertilization.

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

Here, we describe an improvement of the semi-in vitro (SIV) method for observing pollen tube guidance and reception in Arabidopsis thaliana,&#160;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.

In flowering plants, the growth and guidance of the pollen tube (male gametophyte) within the pistil and the reception of the pollen tube by the female ...

An Improved Method to Measure Pollen Hydration Profiles in Arabidopsis thaliana

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

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is established between the interacting partners. Studies across a range of species have revealed that a series of molecular checkpoints regulate the pollen-stigma interaction to ensure that only compatible, generally intraspecific pollen is successful in effecting fertilization. In species that possess a 'dry stigma', such as the model plant Arabidopsis thaliana, the first post-pollination, prezygotic compatibility checkpoint is the establishment of pollen hydration. 
This phase of pollination is tightly regulated, whereby signals from the pollen grain elicit the release of water from the stigma, thus permitting pollen hydration. The ability to accurately measure and track pollen hydration over time is key to the design of experiments directed at understanding the regulation of this critical step in reproduction. Published protocols frequently utilize flowers that have been excised from the parent plant, maintained on liquid or solid media, and bulk pollinated. 
This paper describes a noninvasive, in vivo pollination bioassay that permits minute-by-minute hydration tracking of individual A. thaliana pollen grains at high resolution. The assay is highly reproducible, able to detect very subtle variations of pollen hydration profiles, and thus is suitable for the analysis of mutants that affect pathways regulating pollination. Although the protocol is lengthier than those described for bulk pollinations, the precision and reproducibility it provides, along with its in vivo nature, make it ideal for the detailed dissection of pollination phenotypes.

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

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.

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is ...

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

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Chapters in this video

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

A Method for Characterizing Embryogenesis in Arabidopsis

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

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

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

In Vivo Visualization of Calcium Transients during Fertilization and Early Development in C. elegans

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

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

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

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