Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Summary

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

Abstract

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals.

We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.

Introduction

There are approximately 87,000 chemicals registered for commerce in the United States, yet only a small number of these have been tested for potential health effects¹. Of those that have been tested, only a portion has been assessed for reproductive health effects due in part to the difficulty in determining alteration of the early reproductive events in mammals, especially during female germ cell development and differentiation. Indeed, the first meiotic events take place during the early stages of embryonic development in female mammals and are therefore difficult to access and collect in numbers suitable for screening purposes.

Protocol

1. Preparation of Feeding Bacteria

NOTE: This section describes the preparation of feeding bacteria (E. coli strain OP50).

1. Isolate a single colony of E. coli strain OP50 from a lysogeny broth (LB) agar plate and aseptically inoculate into 300 ml of autoclaved LB broth.
2. Allow the inoculated culture to grow overnight in a shaker at 200 rpm and 37 °C, until saturation is reached.
3. Transfer the OP50 into 6 sterile, pre-weighed 50 ml conical tubes. Pellet the bacteria by centrifuging for 5 min at 6,000 rpm (5,000 x g).
4. Discard the supernatant and wash the bacteria with 50 ml of ster....

Results

Exposure of the Pxol-1::GFP reporter strain to chemical agents such as the microtubule poison Nocodazole (Figure 1) leads to the induction of a high proportion of GFP-expressing embryos in the uterus of exposed adult hermaphrodites compared to DMSO control. The GFP-positive embryos are significantly brighter than the weak background fluorescence observed in other embryos as well as the auto-fluorescence observed in the gut of the animals. Exposed worms are directly imaged on 384-well plates and .......

Discussion

The method described here constitutes the first large scale strategy for the identification of germline toxicants. It requires the use of a GFP transgenic Pxol-1::GFP containing strain that faithfully reports the induction of aneuploidy in early embryos which is used as a proxy for germline dysfunction. The method involves the careful synchronization of a C. elegans worm population and worms’ exposure to chemicals in 96-well format followed by imaging of the GFP positive worms by automated high-co.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
60 mm Vented, sharp edge Petri dishes	Tritech	T3315
Agar	Apex	20-274
Axygen 96-well clear round bottom 2 ml polypropylene deep well plate	Corning	P-DW-20-C-S
Bactopeptone	Apex	20-261
Bacto-tryptone	Fisher Scientific	BP1421-2
Bleach	Clorox
Calcium chloride (CaCl2)	VWR	AA12316-A1
Cholesterol	Fisher Scientific	ICN10138225
DMSO	VWR	IC19018680
E. coli (OP50)	Caenorhabditis Genetics Center		http://www.cbs.umn.edu/cgc
Ethanol 200 proof	VWR	EM-4455S
Greiner CELLSTAR 384 well plates	Sigma-Aldrich	M1937-32EA
ImageXpress Micro XLS System	Molecular Devices
Levamisole hydrochloride	Fluka	31742
Magnesium sulfate anhydrous (MgSO4)	VWR	97061-438
MetaXpress High Content Image Acquisition & Analysis Software	Molecular Devices
Potassium phosphate monobasic (KH2PO4)	Sigma-Aldrich	P5655
Rayon films for biological cultures	VWR	60941-086
Sodium chloride (NaCl)	Sigma-Aldrich	S5886
Sodium hydroxide (NaOH)	Fisher Scientific	S318
Sodium phosphate dibasic (Na2HPO4)	VWR	BDH0316
Stereomicroscope	Nikon	SMZ 745	This microscope has a total magnification form 3.35X to 300X. Any microscope with similar characteristics will work.
TY2441 C. elegans, Pxol-1::GFP reporter strain	Caenorhabditis Genetics Center		http://www.cbs.umn.edu/cgc
Yeast extract	Becton Dickinson	212750