Egg Laying Assay: A Method to Quantify the Egg-Laying Behavior of C. elegans

Abstract

Source: Lee, S. Y. and Kang, K. Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans. J. Vis. Exp. (2017).

C. elegans egg-laying behavior can provide important information in developmental, reproductive, and anatomical studies.  This video introduces the important steps of an egg laying assay and shows a sample experiment in which a drug is tested for reproductive toxicity.

Protocol

Lee and Kang, Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans, J. Vis. Exp. (2017).

1. C. elegans Egg Laying Assay

1. Incubate the age-synchronized eggs on the NGM plate containing DMSO or etoposide for 64 h (before first birth) until the young adult stage at 20 °C.
   NOTE: It is optional to use the worms from the growth measurement experiments. It is convenient to do the growth measurement and egg laying assay together.
2. Transfer 5 adult worms to the new NGM plates without chemicals (condition 1, chemical treatment within a certain period of time, from eggs to young adult stage) or new NGM plate containing the same chemical pretreatment (condition 2, continuous exposure of chemicals through the overall experimental period), and then incubate them for 24 h.
   NOTE: It is recommended to make replicates using 3-5 NGM plates for each treatment; these replicates can be used for statistical analysis.
3. Transfer to the new NGM plate as described above and count the number of eggs every day. Count the worms that crawled off, died, and were internally hatched.
4. Repeat these steps until the worms lay no more eggs, usually in 5 days.
5. Calculate the number of eggs laid per worm from each plate, and sum these values every day for 5 days to determine the total number of eggs laid.
   NOTE: Exclude the number of worms that crawled off and internally hatched from the calculation.

Materials

Caenorhabditis elegans N2	Caenorhabditis Genetics Center (CGC)		Wild type
Dimethyl sulfoxide	Sigma, USA	D2650
Escherichia coli OP50	Caenorhabditis Genetics Center (CGC)
Etoposide	Sigma, USA	E1383
35 × 10 mm Petri dish	SPL Life Sciences, South Korea	10035
Stereo microscope	Nikon, Japan	SMZ800N