Abstract
Fertilization calcium waves are a conserved trigger for animal development; however, genetic analysis of these waves has been limited due to the difficulty of imaging in vivo fertilization. Here we describe a protocol to image calcium dynamics during in vivo fertilization in the genetic animal model Caenorhabditis elegans. This protocol consists of germline microinjection of a chemical calcium indicator, worm immobilization, live imaging, and image processing that quantifies the calcium fluorescence in the oocyte region moving in the field-of-view during ovulation. This imaging protocol can also be used to image other cellular processes during in vivo fertilization in C. elegans, such as membrane fusion and cytoskeletal dynamics.
Keywords: Fertilization, Fertilization calcium waves, Calcium waves, Caenorhabditis elegans, Live imaging, Calcium imaging, Image processing
Background
Fertilization calcium waves play a pivotal role in egg activation and have been analyzed in various organisms by using in vitro fertilization systems. The nematode Caenorhabditis elegans is amenable to imaging in vivo fertilization because of its translucent body. The fertilization calcium imaging in C. elegans by using a chemical calcium indicator is reported in Samuel et al. (2001). We describe here a protocol modified from the imaging method by applying high-speed spinning disk confocal microscopy and image processing methods that segment the fertilized oocyte region moving in the field-of-view during ovulation. This protocol enables a precise quantitative description of the temporal dynamics of the calcium waves and genetic analysis of the wave pattern.
Materials and Reagents
Equipment
Software
Procedure
Note: This protocol is modified from the original method described in Samuel et al. (2001).
Data analysis
Notes
The worm immobilization protocol can be used to image other cellular processes during fertilization such as membrane fusion or cytoskeleton dynamics. For membrane fusion imaging, use the OD58 [oocyte GFP::PH] strain (Audhya et al., 2005) and males of EG4883 [sperm mCherry::histone] (Frøkjaer-Jensen et al., 2008) or the ONA18 [sperm TRP-3::tagRFP-T] strain (Takayama and Onami, 2016) instead of the wild-type N2. For filamentous actin imaging, use the BV67 [lifeact::gfp] strain (Pohl and Bao, 2010).
Recipes
Acknowledgments
The protocol is used in Takayama and Onami (2016). The protocol for the microinjection of a chemical calcium indicator dye is based on that described by Samuel et al. (2001). The immobilization method is based on the method of Kim et al. (2013). The uneven illumination correction is derived from the work of Wolf et al. (2007). The Calculator_Plus3 plugin was modified from the Calculator_Plus plugin, which was developed by Wayne Rasband and Gabriel Landini. Portions of the ContourTrack3Stack_, ContourTrack4Stack_, and TrackOocyte_ plugin codes were reused from a program written by Takuya Maeda. We thank Asako Sugimoto for her advice regarding the choice of microscopy filters. We also thank Koji Kyoda and Rie Furushima for their assistance. This work was supported, in part, by the National Bioscience Database Center (NBDC) of the Japan Science and Technology Agency (S.O.), by the Strategic Programs for R&D (President’s Discretionary Fund) of RIKEN (S.O.), and by JSPS KAKENHI Grant Number 15K18547 (J.T.). The authors declare that they have no competing interests.
References
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