Name: In Vivo Visualization of Calcium Transients during Fertilization and Early Development in C. elegans (Video) | JoVE Journal
Uploaded: 2024-07-12T15:00:00.000+00:00
Duration: 5 min 11 s
Description: Learn to visualize calcium transients during fertilization and early development in C. elegans using the CaFE reporter. This method reveals delayed calcium waves and single-cell dynamics with standard microscopy equipment.

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5 minutes

KSKG was funded by a grant from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (R15HD111986). Some strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD4010440). We thank WormBase.

Tracking Calcium in the Early Developmental Phases of C. elegans

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<li>McAvey, B. A., Wortzman, G. B., Williams, C. J., Evans, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Involvement+of+calcium+signaling+and+the+actin+cytoskeleton+in+the+membrane+block+to+polyspermy+in+mouse+eggs%5BTitle%5D)+AND+%22Biolo+Reprod%22%5BJournal%5D)">Involvement of calcium signaling and the actin cytoskeleton in the membrane block to polyspermy in mouse eggs.</a> Biolo Reprod. 67 (4), 1342-1352 (2002).</li>
<li>Takayama, J., Onami, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+sperm+TRP-3+channel+mediates+the+onset+of+a+Ca+2%2B+wave+in+the+fertilized+C.+elegans+oocyte%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">The sperm TRP-3 channel mediates the onset of a Ca 2+ wave in the fertilized C. elegans oocyte.</a> Cell Rep. 15 (3), 625-637 (2016).</li>
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<li>Samuel, A. D., Murthy, V. N., Hengartner, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcium+dynamics+during+fertilization+in+C.+elegans%5BTitle%5D)+AND+%22BMC+Dev+Biol%22%5BJournal%5D)">Calcium dynamics during fertilization in C. elegans.</a> BMC Dev Biol. 1 (1), 8(2001).</li>
<li>Dana, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-performance+calcium+sensors+for+imaging+activity+in+neuronal+populations+and+microcompartments%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">High-performance calcium sensors for imaging activity in neuronal populations and microcompartments.</a> Nat Methods. 16 (7), 649-657 (2019).</li>
<li>Golden, N. L., Plagens, R. N., Kim Guisbert, K. S., Guisbert, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Standardized+methods+for+measuring+induction+of+the+heat+shock+response+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Standardized methods for measuring induction of the heat shock response in Caenorhabditis elegans.</a> J Vis Exp. (161), e61030(2020).</li>
<li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fiji%3A+an+open-source+platform+for+biological-image+analysis%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Fiji: an open-source platform for biological-image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</li>
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<li>Kachur, T. M., Audhya, A., Pilgrim, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((UNC-45+is+required+for+NMY-2+contractile+function+in+early+embryonic+polarity+establishment+and+germline+cellularization+in+C.+elegans%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">UNC-45 is required for NMY-2 contractile function in early embryonic polarity establishment and germline cellularization in C. elegans.</a> Dev Biol. 314 (2), 287-299 (2008).</li>
<li>Takayama, J., Fujita, M., Onami, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+live+imaging+of+calcium+waves+and+other+cellular+processes+during+fertilization+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Bio+Protoc%22%5BJournal%5D)">In vivo live imaging of calcium waves and other cellular processes during fertilization in Caenorhabditis elegans.</a> Bio Protoc. 7 (7), e2205(2017).</li>
<li>Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., Mello, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Potent+and+specific+genetic+interference+by+double-stranded+RNA+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.</a> Nature. 391 (6669), 806-811 (1998).</li>
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<li>Kamath, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Systematic+functional+analysis+of+the+Caenorhabditis+elegans+genome+using+RNAi%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Systematic functional analysis of the Caenorhabditis elegans genome using RNAi.</a> Nature. 421 (6920), 231-237 (2003).</li>
<li>Reboul, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((elegans+ORFeome+version+1.1%3A+experimental+verification+of+the+genome+annotation+and+resource+for+proteome-scale+protein+expression%5BTitle%5D)+AND+%22Nat+Genet%22%5BJournal%5D)">elegans ORFeome version 1.1: experimental verification of the genome annotation and resource for proteome-scale protein expression.</a> Nat Genet. 34 (1), 35-41 (2003).</li>
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<li>Paudel, S., Yue, M., Nalamalapu, R., Saha, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deciphering+the+calcium+code%3A+A+review+of+calcium+activity+analysis+methods+employed+to+identify+meaningful+activity+in+early+neural+development%5BTitle%5D)+AND+%22Biomolecules%22%5BJournal%5D)">Deciphering the calcium code: A review of calcium activity analysis methods employed to identify meaningful activity in early neural development.</a> Biomolecules. 14 (1), 138(2024).</li>
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<li>Johnston, W. L., Dennis, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+eggshell+in+the+C.+elegans+oocyte-to-embryo+transition%5BTitle%5D)+AND+%22Genesis%22%5BJournal%5D)">The eggshell in the C. elegans oocyte-to-embryo transition.</a> Genesis. 50 (4), 333-349 (2012).</li>
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<li>Zacharias, A. L., Walton, T., Preston, E., Murray, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+differences+in+nuclear+%CE%B2-catenin+and+TCF+pattern+embryonic+cells+in+C.+elegans%5BTitle%5D)+AND+%22PLoS+Genet%22%5BJournal%5D)">Quantitative differences in nuclear β-catenin and TCF pattern embryonic cells in C. elegans.</a> PLoS Genet. 11 (10), e1005585(2015).</li>
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The authors declare no competing or financial interests.

A simple tool with a robust protocol is a potent combination to tackle difficult scientific questions. Here, methods for the visualization of calcium as an easily detectable proxy for fertilization are presented using the CaFE reporter. This same reporter persists into early embryogenesis and also allows the visualization of calcium transients further into development. Calcium signaling serves as a critical second messenger that demarcates major shifts in cellular function, particularly for developmental biology. In the oocyte, the first burst phase of the&#160;calcium wave marks&#160;not just the timing of fertilization but also the site of sperm&#160;fusion3. In C. elegans, the A-P axis is also determined by the site of sperm fusion23. Therefore, the ability to visualize calcium in oocytes and embryos allows for the investigation of complex questions that are central to cell and developmental biology.
The method described here with the CaFE reporter should be straightforward enough for nematode novices. Previous methods to detect calcium waves in oocytes of C. elegans relied upon dye injection3,8,14. While these studies were important and illuminating, dye injection is labor-intensive, and injection equipment is not available in every lab.
The protocols presented here have been optimized for healthy worms. To maximize the chance for success, ensure that there is no contamination on the worm media or in the bacteria lawn on which the worms feed. Do not expose the worms to stress conditions, such as temperature, as these will affect ovulation and fertilization. Additionally, controls for RNAi efficacy should be included with every experiment as efficacy decreases over time. Use a positive control for RNAi efficacy like egg-5,&#160;which generates embryos, but no viable progeny as eggshell formation is compromised24. Furthermore, the image capture parameters must be optimized for each microscopy system. Our specifications are included here as a reference, but deviations are to be expected. Although this system readily detects cytoplasmic calcium as a proxy for fertilization, it does not necessarily represent a bona fide sperm-oocyte fusion event.
The wide array of tools and mutants freely distributed within the worm community enhance the utility of the CaFE reporter. The reporter is integrated into the worm genome and is easily crossed into other mutant or reporter C. elegans strains25. Reported here is the creation of EAG25 expressing the CaFE reporter with a histone H2B-mCherry marker to visualize nuclei (Figure 1) and EAG28 with both the CaFE reporter and a pleckstrin-homology domain-mCherry marker, which highlights the cell periphery (Figure 2)13,26. Both strains aid in the visualization of cells in the germline and during embryogenesis. Furthermore, the facility of RNAi in 
C. elegans, when used with the CaFE reporter, have revealed new&#160;insights into fertilization competence. As shown in the double ovulation induced by ipp-5 RNAi knockdown in Figure 3, the presence of sperm and an ovulated oocyte is insufficient to stimulate a fertilization event.
These results indicate that another signal, or the absence of an inhibitor, must exist that allows the oocyte to become fertilized. The prematurely ovulated oocyte displays a delayed calcium transient when the oocyte moves into the uterus. This late calcium wave suggests that the prematurely ovulated -2 oocyte can develop fertilization competency with time. We anticipate that studies on the timing of fertilization, particularly with regard to cell-cell signaling and regulation, will be aided by the use of the CaFE reporter. Additionally, the CaFE reporter persists into embryogenesis and displays single-cell calcium transients. This embryonic calcium signaling was also reported within the first 24 hours of zebrafish development27. The role of the calcium transients is unknown, but the presence suggests a cell signaling event during development that has not yet been explored. Notably, the calcium transients were not observed in newly fertilized zygotes. Therefore, calcium does not localize to the cleavage furrow as has been documented in several other organisms, including Xenopus and humans20,21,22.
Importantly, the CaFE reporter is easy enough to be used by undergraduates with minimal training. We have designed and executed a 1-credit CURE (course-based undergraduate research experience) lab for biology students with the strains and protocols described here. Over the course of a semester, the class met once a week for 3 h or twice a week for 1 h 30 min each. Students were given the choice to work by themselves or in groups of 2. Each student/pair selected a different gene to study from a curated list. They performed RNAi against their chosen gene in the&#160;EAG28 strain and examined worms for effects on fecundity,&#160;fertilization, and/or gonad morphology. Based on their results and their background reading using primary literature, the students developed hypotheses that they could test in subsequent experiments. This iterative design was critical to increasing student engagement28. The students obtained authentic research experience and gained skills in model organisms, genetic screens, and fluorescence microscopy. Given the ease of use of the CaFE reporter, students with no research experience were able to succeed. Afterward, the students overwhelmingly expressed a preference for the CURE format over traditional lab classes, with many students expressing a desire to continue with research. Taken together, these tools and protocols aid in both education and research into early developmental processes.

Fertilization marks the beginning of a new life cycle, but defining the precise moment of fertilization is challenging. A conserved feature of fertilization is a wave of calcium across the oocyte immediately after sperm fusion1. Although the nature of the calcium wave, in terms of frequency and speed, differs across species, nearly all organisms exhibit a transitory increase in intracellular calcium after fertilization. The calcium wave plays a critical role in a block to polyspermy, egg activation, and other important cellular events2. Since the calcium wave initiates at the site of sperm fusion, calcium serves as a marker for fertilization3.
Caenorhabditis elegans is an ideal model organism for studying early development. The worms are transparent hermaphrodites that are genetically tractable4. Most importantly for the study of fertilization, C. elegans adults continuously produce oocytes using a strictly ordered gonad5. In fact, oocytes are the only new cells produced as the soma is post-mitotic in adulthood6. Figure 1 highlights the gonad, which consists of 2 symmetrical U-shaped tubes of developing oocytes. The oocyte closest to the spermatheca (storage organ for sperm) is termed the -1 oocyte. This assembly-line design of the worm gonad ovulates a mature oocyte into the spermatheca every ~23 min at 20 &#176;C in young adults7. The newly fertilized embryo then moves into a shared uterus before being laid through a single vulva.
Previous techniques to visualize calcium during fertilization in C. elegans relied upon microinjection of calcium-sensitive dyes3,8. To more easily view the calcium wave upon sperm-egg fusion, a genetically encoded calcium indicator based on jGCaMP7s was inserted in single-copy at a safe harbor locus using clustered regularly interspaced short palindromic repeats (CRISPR)7,9. The reporter was termed CaFE for Calcium during Fertilization in C. elegans. The reporter shows no significant defects in physiology or fecundity.
Here, protocols are presented for visualization of the calcium wave in the oocytes and embryos of C. elegans using the CaFE reporter. Combined with the myriad tools available in the worm community, such as RNA interference (RNAi) and mCherry gonad markers, the CaFE reporter facilitates investigation into the regulation of internal fertilization events, particularly fertilization competence and the timing of fertilization. Additionally, the CaFE reporter persists into early development and is a unique tool to probe embryogenesis.

<table><tbody><tr><td>Agar</td><td>Fisher Scientific</td><td>DF0140-07-4</td><td>2 kg; Powder dissolves easier than flakes</td></tr><tr><td>Agarose</td><td>MidSci</td><td>BE-A125</td><td>500 g</td></tr><tr><td>Alcohol lamp</td><td>Fisher Scientific</td><td>S13475</td><td>Use with 95% ethanol</td></tr><tr><td>Ammonium chloride (NH4Cl)</td><td>Fisher Scientific</td><td>AAA1500030</td><td>250 g</td></tr><tr><td>Ampicillin</td><td>Fisher Scientific</td><td>BP1760-5</td><td>5 g</td></tr><tr><td>AMSCO 400 Series Small Steam Sterilizer</td><td>Steris Healthcare</td><td>N/A</td><td></td></tr><tr><td>Bacto-peptone</td><td>Fisher Scientific</td><td>BP1420-500</td><td>500 g</td></tr><tr><td>Bacto-tryptone</td><td>Fisher Scientific</td><td>DF0123-17-3</td><td>500 g</td></tr><tr><td>Calcium chloride (CaCl2)</td><td>Fisher Scientific</td><td>C69-500</td><td>500 g</td></tr><tr><td>Cholesterol</td><td>Thermo Scientific</td><td>A11470.18</td><td>50 g</td></tr><tr><td>Dragonfly 200 spinning disk confocal</td><td>Oxford Instruments Andor</td><td>N/A</td><td>Used with Leica microscope</td></tr><tr><td>Fisherbrand Superfrost Cytogenics Microscope Slides</td><td>Fisher Scientific</td><td>22-035-900</td><td>144 slides per pack</td></tr><tr><td>Flat Nose Pliers, Smooth Jaw</td><td>Home Depot</td><td>305530604</td><td>Ensure pliers are smooth jaw</td></tr><tr><td>Isopropyl &amp;beta;- d-1-thiogalactopyranoside&amp;nbsp;(IPTG)</td><td>Fisher Scientific</td><td>BP1755-10</td><td>10 g; dioxane-free</td></tr><tr><td>Laboratory tape</td><td>Fisher Scientific</td><td>15-901-10R</td><td>0.5 inch tape is used to tape microscope slides</td></tr><tr><td>Levamisole</td><td>Fisher Scientific</td><td>AC187870100</td><td>10 g</td></tr><tr><td>Magnesium sulfate (MgSO4)</td><td>Fisher Scientific</td><td>M63-500</td><td>500 g</td></tr><tr><td>Microscope cover glass</td><td>Fisher Scientific</td><td>12541016</td><td>1 oz pack</td></tr><tr><td>Nikon ECLIPSE Ti2 laser scanning confocal</td><td>Nikon</td><td>N/A</td><td></td></tr><tr><td>Nikon NIS Elements software</td><td>Nikon</td><td>N/A</td><td>Confocal</td></tr><tr><td>OP50 Escherichia coli</td><td>Caenorhabditis Genetics Center (CGC)</td><td>OP50</td><td></td></tr><tr><td>Platinum Wire</td><td>TriTech</td><td>PT-9010</td><td></td></tr><tr><td>Potassium phosphate dibasic (K2HPO4)</td><td>Fisher Scientific</td><td>P288-500</td><td>500 g</td></tr><tr><td>Potassium phosphate monobasic (KH2PO4)</td><td>Fisher Scientific</td><td>AA1159436</td><td>500 g</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Fisher Scientific</td><td>S271-500</td><td>500 g</td></tr><tr><td>Sodium phosphate dibasic heptahydrate (Na2HPO4)</td><td>Fisher Scientific</td><td>S471-3</td><td>3 kg</td></tr><tr><td>Stereo microscope</td><td>Leica</td><td>KL300 LED</td><td></td></tr><tr><td>Sterile Petri dish (35 mm x 10 mm)</td><td>CellTreat</td><td>229638</td><td>960 Petri dishes per case</td></tr><tr><td>Sterile Petri dish (60 mm x 15 mm)</td><td>CellTreat</td><td>229665</td><td>500 Petri dishes per case</td></tr><tr><td>Strain EAG16 &lt;em&gt;spn-4p::jGCaMP7s::pie-1u&lt;/em&gt;</td><td>Caenorhabditis Genetics Center (CGC)</td><td>EAG16</td><td>Created by Kim Guisbert Lab</td></tr><tr><td>Strain EAG25 &lt;em&gt;spn-4p::jGCaMP7s::pie-1u; ujIs113 II.&lt;/em&gt;</td><td>Caenorhabditis Genetics Center (CGC)</td><td>EAG25</td><td>Created by Kim Guisbert Lab</td></tr><tr><td>Strain EAG28 &lt;em&gt;spn-4p::jGCaMP7s::pie-1u; unc-119(ed3) III; ltIs44 V.&lt;/em&gt;</td><td>Caenorhabditis Genetics Center (CGC)</td><td>EAG28</td><td>Created by Kim Guisbert Lab</td></tr><tr><td>Strain JIM113 &lt;em&gt;ujIs113 II [pie-1p::mCherry::H2B::pie-1 3&#039;UTR + nhr-2p::his-24::mCherry::let-858 3&#039;UTR + unc-119(+)]&lt;/em&gt;</td><td>Caenorhabditis Genetics Center (CGC)</td><td>JIM113</td><td>Created by E. Preston - Murray Lab</td></tr><tr><td>Strain OD70 &lt;em&gt;unc-119(ed3) III; ltIs44 V [pie-1p::mCherry::PH(PLC1delta1) + unc-119(+)]&lt;/em&gt;</td><td>Caenorhabditis Genetics Center (CGC)</td><td>OD70</td><td>Created by Audhya/Oegema - Greenstein Lab</td></tr><tr><td>Tritech Worm Pick Handle</td><td>TriTech</td><td>TWPH1</td><td></td></tr><tr><td>Yeast extract</td><td>IBI Scientific</td><td>IB49160</td><td>500 g</td></tr></tbody></table>

in vivo visualization of calcium transients during fertilization and early development in 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.

Using the protocols outlined in this manuscript, the dynamic patterns of calcium signaling in fertilization and embryogenesis were observed in C. elegans.
A typical fertilization sequence in worms containing the CaFE reporter is shown in Figure 2. To facilitate analysis, the EAG28 strain was used which combines the CaFE reporter with a pleckstrin homology domain (PH-mCherry) transgene13. The PH-mCherry marker localizes to the plasma membrane and allows for easier visualization of cell boundaries, particularly in the gonad arm. The EAG28 strain was created by crossing the EAG16 strain containing the CaFE reporter with the OD70 PH-mCherry reporter strain13. In C. elegans, ovulation and fertilization occur concurrently. Time 0 of fertilization is considered the first frame where a fluorescence signal is detected in the ovulating oocyte (Figure 2). A bright burst of fluorescence occurs at the site of sperm fusion as soon as the leading edge of the oocyte enters the spermatheca, the storage organ for sperm7. The signal appears before ovulation is complete. The calcium wave is biphasic, with a rapid initial burst followed by a wave of fluorescence from the entry point toward the opposite pole. The entire oocyte becomes fluorescent in &lt;30 s.
Note that the CaFE reporter is in a single copy, and therefore, the signal is not as bright as other transgenes. Z-stack time-lapse is not recommended during ovulation unless a spinning disk confocal is available and optimized. Z stack images can be obtained before or after a movie to capture 3D changes in gonad morphology. Although the images here were taken with a confocal microscope, the transients have also been observed using widefield fluorescence microscopy which is more common and affordable than confocal. Using a laser scanning confocal, the frame rate is approximately 1 fps. Using a spinning disk confocal, the frame rate is 10 fps or faster. The background signal of the CaFE reporter is sufficient to illuminate the maturing oocytes in the gonad arm. Upon fertilization, a 1.5-2x increase in GFP signal intensity is typically observed.
Several alternative methods exist for quantitation. For example, the Imaris image analysis software, created by Oxford Instruments (the same parent company as the Andor Dragonfly spinning disk confocal microscope), has the ability to quantitate the signal in each frame from only the transiting oocyte instead of a bigger ROI. However, the Imaris software is not free. Additionally, a detailed image analysis strategy to measure calcium waves in the oocyte has been described in excellent detail by Takayama, Fujita, and Onami and uses ImageJ, which is freely available14. Alternatively, kymographs to illustrate the wave of fluorescent signal across a single oocyte require more extensive image manipulation than described here but are described in Takayama and Onami3.
A distinct advantage of C. elegans is its ability to knockdown the expression of nearly any gene by feeding the worms bacteria expressing dsRNA to trigger endogenous RNAi15,16. For optimal dsRNA expression, both the RNAi plates and the saturated RNAi culture contain IPTG. The dsRNA is produced by expressing the gene of interest with flanking promoters. Here, we have used the Ahringer library which targets most of the worm genome through the use of 16,256 bacterial strains17. Alternatively, an ORFeome of ~11,000 RNAi clones was created by the Vidal lab using the Gateway system and is available for purchase through Horizon Discovery18.
To demonstrate the utility of the CaFE reporter in combination with RNAi, we examined premature ovulation. A partial deletion in ipp-5 induces a double ovulation event where both the -1 oocyte and the -2 oocyte enter the spermatheca during the same ovulation19. IPP-5 is a phosphatase that acts on IP3 and effectively decreases levels of the IP3 second messenger. We found that RNAi knockdown of ipp-5 elicits a double ovulation phenotype similar to the mutant.
Analysis of the CaFE reporter during the double ovulation induced by ipp-5 RNAi knockdown revealed novel aspects of the OET. First, a calcium signal was observed immediately upon entry into the spermatheca in the leading -1 oocyte but not in the trailing -2 oocyte (Figure 3). Although 2 oocytes are ovulated, the -1 oocyte is still the only oocyte that shows signs of proper maturation, in particular, the breakdown of the nuclear envelope (NEBD). These data suggest that even though the -2 oocyte is in the presence of sperm, it is not competent to be fertilized as it has not yet properly matured. Second, the -2 oocyte exhibits a delayed calcium wave, typically when the oocyte exits the spermatheca for the uterus. Although oocyte maturation is normally a prerequisite for ovulation, these data suggest that the maturation of the oocyte and competence for fertilization can still occur after ovulation. The visualization of the delayed calcium wave during ipp-5 knockdown highlights the advantages of the CaFE reporter to interrogate fertilization competence and timing.
Furthermore, we have discovered that the CaFE reporter is detectable in laid eggs and reveals single-cell calcium transients during early embryogenesis in C. elegans. Embryos from synchronized Day 1 adult EAG28 (CaFE; PH-mCherry) worms were imaged using the protocols from sections 1-5 with the Andor Dragonfly spinning disk confocal using the Zyla camera (Figure 4). The calcium transients are confined to single cells and take less time than the fertilization calcium wave to complete (~9 s, n=11). However, the calcium wave during embryogenesis does not appear to be biphasic. Calcium transients were not observed before the 8-cell stage. Notably, calcium does not localize to the cleavage furrow in C. elegans, as is seen in many other organisms, including humans and Xenopus20,21,22. Single-cell calcium transients were observed well after gastrulation (~200 min after fertilization) and in laid eggs. A single embryo displays multiple calcium transients over time but in different cells and typically one cell at a time. Note that continuous exposure to laser stimulation will damage the embryos. Either lower LED/laser power or discontinuous exposure is recommended. However, since the calcium waves are relatively short at ~9 s, we do not recommend slower than 2 s between LED/laser stimulation events.
<img alt="Caenorhabditis elegans reproduction; fluorescence microscope image, labeled gonad, oocytes, embryos." class="xfigimg" src="/files/ftp_upload/67141/67141fig01.jpg" data-altupdatedat="1788840098000" data-alt="Figure 1"> 
Figure 1: C. elegans gonad arm. Image of one gonad arm from the EAG25 worm strain, showing the CaFE reporter GFP (green) and the histone 2B-mCherry marker (red), which highlights nuclei in the germline. The proximal oocyte is denoted by the -1 directly before the spermatheca, where the sperm are stored (arrow). The embryo located closest to the spermatheca but inside the uterus is the most recent embryo. The image was taken using the Nikon ECLIPSE Ti2 laser scanning confocal. Scale bar = 20 µm. <a href="https://www.jove.com/files/ftp_upload/67141/67141fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Time-lapse microscopy of cellular process; fluorescence imaging, cell dynamics analysis." class="xfigimg" src="/files/ftp_upload/67141/67141fig02.jpg" data-altupdatedat="1788840098000" data-alt="Figure 2"> 
Figure 2: Visualization of the calcium wave during fertilization. Time series images of fluorescence reflecting intracellular calcium during fertilization in the EAG28 worm strain. The EAG28 strain contains both the CaFE reporter (green) and a pleckstrin-homology domain-mCherry fusion (yellow), which highlights plasma membranes. Time 0 indicates the first frame portraying a clear increase in fluorescent signal in the oocyte. Time 24.4 s reflects the first frame showing whole oocyte fluorescence. Time series images were taken using the Andor Dragonfly spinning disk confocal with a Zyla camera. Scale bar = 20 µm. <a href="https://www.jove.com/files/ftp_upload/67141/67141fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Fluorescence time-lapse showing protein migration in cells, microscopy, cellular dynamics analysis." class="xfigimg" src="/files/ftp_upload/67141/67141fig03.jpg" data-altupdatedat="1788840098000" data-alt="Figure 3"> 
Figure 3: Calcium waves during an ipp-5 RNAi-induced double ovulation event. Time-lapse images of sequential calcium waves of the -1 oocyte and -2 oocyte in an EAG16 strain (CaFE in GFP green) exposed to ipp-5 RNAi. The -40.0 s frame includes labels for the -1 oocyte (-1), -2 oocyte (-2), spermatheca (*), and +1 embryo (+1). Time 0 s displays the initial increase in fluorescence of the calcium wave of the -1 oocyte. Time 4.5 s shows the calcium wave spreading towards the opposite pole in the -1 oocyte. Time 70.5 s displays whole cell fluorescence in the -1 oocyte accompanied by the -2 oocyte at baseline GFP signal; both oocytes are in the spermatheca. The fertilized -1 oocyte and unfertilized -2 oocyte enter the uterus at time 158.3 s. At 219.3 s, the -2 oocyte displays a late fluorescent calcium wave. Images were taken using the Andor Dragonfly spinning disk confocal with a Zyla camera. Scale bar = 30 µM <a href="https://www.jove.com/files/ftp_upload/67141/67141fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Fluorescence microscopy, plant cells, chlorophyll, green fluorescence, cellular structure analysis." class="xfigimg" src="/files/ftp_upload/67141/67141fig04.jpg" data-altupdatedat="1788840098000" data-alt="Figure 4"> 
Figure 4: Single-cell calcium transients in early embryogenesis. (A) Embryos in utero and (B) ex utero (laid egg) displayed a fluorescent signal in a single cell during embryogenesis. Images were taken using the Andor Dragonfly spinning disk confocal with a Zyla camera. Scale bar = 20 µm. <a href="https://www.jove.com/files/ftp_upload/67141/67141fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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JoVE Journal

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