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>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

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

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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.

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Live Confocal Imaging of Developing Arabidopsis Flowers

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Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers.

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

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to laboratories for studies to further our understanding of marine mammal reproduction. Heterologous IVF, a replacement for homologous IVF, could provide a means to test the sperm fertility potential; to study gamete physiology and early embryo development; and to avoid the use of valuable dolphin oocytes, which are difficult to obtain. Here, we present protocols that have been successfully used to collect and cryopreserve dolphin spermatozoa. The collection of semen is performed by manual stimulation on trained dolphins. Cryopreservation is accomplished using a TRIS egg-yolk based extender with glycerol. In addition, we present a protocol that describes heterologous IVF using dolphin spermatozoa and bovine oocytes and that verifies the hybrid nature of the resulting embryo using PCR. Heterologous fertilization raises questions on fertilization and can be used as a tool to study gamete physiology and early embryo development. In addition, the success of heterologous IVF demonstrates the potential of this technique to test dolphin sperm fertilizing capacity, which is worth further examination.

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

Here, we present protocols that have been successfully used for dolphin spermatozoa collection, cryopreservation, and heterologous IVF performance using bovine oocytes.

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

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previously developed in mice. Specifically, the chromosomes and the centromeres of metaphase II (MII) oocytes were fluorescently labeled and counted on sequential focal plans after confocal laser microscope scanning. This analysis revealed a higher incidence in the aneuploidy rate when immature oocytes were collected from the follicles and matured in vitro compared to in vivo. Immunostaining for tubulin and the acetylated form of histone four at specific lysine residues also revealed differences in the morphology of the meiotic spindle and in the global pattern of histone acetylation. Finally, the expression of mRNAs coding for histone deacetylases (HDACs) and acetyl-transferases (HATs) was investigated by reverse transcription and quantitative-PCR (q-PCR). No differences in the relative expression of transcripts were observed between in vitro and in vivo matured oocytes. In agreement with a general silencing of the transcriptional activity during oocyte maturation, the analysis of the total transcript amount can only reveal mRNA stability or degradation. Therefore, these findings indicate that other translational and post-translational regulations might be affected.
Overall, the present study describes an experimental approach to morphologically and biochemically characterize the horse oocyte, a cell type that is extremely challenging to study due to low sample availability. However, it can expand our knowledge on the reproductive biology and infertility in monovulatory species.

This manuscript describes an experimental approach to morphologically and biochemically characterize horse oocytes. Specifically, the present work illustrates how to collect immature and mature horse oocytes by ultrasound-guided ovum pick-up (OPU) and how to investigate chromosome segregation, spindle morphology, global histone acetylation, and mRNA expression.

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

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, early stage plant embryos are small and contain few cells, making them difficult to observe and analyze. A method is described here for characterizing pattern formation in plant embryos under a microscope using the model organism Arabidopsis. Following the clearance of fresh ovules using Hoyer's solution, the cell number in and morphology of embryos could be observed, and their developmental stage could be determined by differential interference contrast microscopy using a 100X oil immersion lens. In addition, the expression of specific marker proteins tagged with Green Fluorescent Protein (GFP) was monitored to annotate cell identity specification during embryo patterning by confocal laser scanning microscopy. Thus, this method can be used to observe pattern formation in wild-type plant embryos at the cellular and molecular levels, and to characterize the role of specific genes in embryo patterning by comparing pattern formation in embryos from wild-type plants and embryo-lethal mutants. Therefore, the method can be used to characterize embryogenesis in Arabidopsis.

A Method for Characterizing Embryogenesis 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.

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

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

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

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. The classical histochemical reaction is based on the hydrolysis of the substrate X-gal in combination with ferric and ferrous ions, which produces an insoluble blue precipitate that is easy to visualize. Therefore, &#946;-galactosidase activity serves as a marker for the expression pattern of the gene of interest as the development proceeds. Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the subsequent method for paraffin sectioning and counterstaining. Additionally, a procedure for clarifying whole embryos is provided to better visualize X-gal staining in deeper regions of the embryo. Consistent results are obtained by performing this procedure, although optimization of reaction conditions is needed to minimize background activity. Limitations in the assay should be also considered, particularly regarding the size of the embryo in whole mount staining. Our protocol provides a sensitive and a reliable method for &#946;-galactosidase detection during the mouse development that can be further applied to the cryostat sections as well as whole organs. Thus, the dynamic gene expression patterns throughout development can be easily analyzed by using this protocol in whole embryos, but also detailed expression at the cellular level can be assessed after paraffin sectioning.

Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the method for paraffin sectioning and counterstaining. This is an easy and quick procedure to monitor gene expression during development that can also be applied to tissue sections, organs or cultured cells.

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

Standard spermiograms describing sperm quality are mostly based on the physiological and visual parameters, such as ejaculate volume and concentration, motility and progressive motility, and sperm morphology and viability. However, none of these assessments is good enough to predict the semen quality. Given that maintenance of sperm viability and fertilization potential depends on membrane integrity and intracellular functionality, evaluation of these parameters might enable a better prediction of sperm fertilization competence. Here, we describe three feasible methods to evaluate sperm quality using specific fluorescent probes combined with fluorescence microscopy or flow cytometry analyses. Analyses assessed plasma membrane integrity using 4&#39;,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI), acrosomal membrane integrity using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and mitochondrial membrane integrity using 5,5&#39;,6,6&#39;-tetra-chloro-1,1&#39;,3,3&#39;-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). Combinations of these methods are also presented. For instance, use of annexin V combined with PI fluorochromes enables assessing apoptosis and calculating the proportion of apoptotic sperm (apoptotic index). We believe that these methodologies, which are based on examining spermatozoon membranes, are very useful for the evaluation of sperm quality.

Here, we present methodologies to evaluate spermatozoan membrane integrity, a cellular feature associated with sperm fertilization competence. We describe three techniques for the fluorimetric assessment of sperm membranes: simultaneous staining with specific fluorescent probes, fluorescence microscopy, and advanced sperm-dedicated flow cytometry. Examples of combining the methodologies are also presented.

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

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

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

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 longevity can be a powerful way to reveal mechanisms of tissue development and maintenance. One of the most important tools currently available to monitor cells in vivo is the Confetti mouse model. The Confetti model can be used to genetically label individual cells in living mice with various fluorescent proteins in a cell type-specific manner and monitor their fate, as well as the fate of their progeny over time, in a process called clonal genetic tracing or clonal lineage tracing. This model was generated almost a decade ago and has contributed to an improved understanding of many biological processes, particularly related to stem cell biology, development, and renewal of adult tissues. However, preserving the fluorescent signal until image collection and simultaneous capturing of various fluorescent signals is technically challenging, particularly for mineralized tissue. This publication describes a step-by-step protocol for using the Confetti model to analyze growth plate cartilage that can be applied to any mineralized or nonmineralized tissue.

This method describes the use of the R26R-Confetti (Confetti) mouse model to study mineralized tissues, covering all steps from the breeding strategy to the image acquirements. Included is a general protocol that can be applied to all soft tissues and a modified protocol that can be applied to mineralized tissues.

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 ...

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

Live Confocal Imaging of Developing Arabidopsis Flowers

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

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

A Method for Characterizing Embryogenesis in Arabidopsis

Live Cell Imaging of Chromosome Segregation During Mitosis

Tracing Gene Expression Through Detection of β-galactosidase Activity in Whole Mouse Embryos

Fluorimetric Techniques for the Assessment of Sperm Membranes

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

Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues