Name: comprehensive assessment of germline chemical toxicity using the nematode caenorhabditis elegans
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Description: Watch this Scientific Journal Video about Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans at JoVE.com

The authors would like to thank the following funding sources NIH ES020353 and the Colgate-Palmolive Alternative Research Grant award for making this work possible.

1. Preparation of Feeding Bacteria
NOTE: This section describes the preparation of feeding bacteria (E. coli strain OP50).
<ol>
	<li>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.</li>
	<li>Allow the inoculated culture to grow overnight in a shaker at 200 rpm and 37 &#176;C, until saturation is reached.</li>
	<li>Transfer the OP50 into 6 sterile, pre-weighed 50 ml conical tub...

<ol>
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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&#8217; exposure to chemicals in 96-well format followed by imaging of the GFP positive worms by automated high-co...

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.
The germline provides the crucial link between generations, and its appropriate function is dependent upon the precise execution of the intricate program of cellular and chromosomal division termed meiosis. Dysregulation of the meiotic process may result in reduced fertility and the production of gametes and embryos with an abnormal number of chromosomes, a condition termed aneuploidy. Chromosome segregation errors in meiosis are highly relevant to human health. Chromosome abnormalities are common, with a frequency of 1 in 150 live births, Trisomy 21, 18 and 13 as well as X and Y chromosome errors being the most prevalent types2,3. Furthermore, congenital malformations, including those of chromosomal origins, are the leading cause of infant death in the U.S.4 The idea that environmental influences can affect chromosomal segregation and behavior is not new5, but is still poorly understood. It is therefore crucial to investigate which of the chemicals introduced into our environment are interfering with human fertility, early development and overall reproductive health.
In light of these limitations of the mammalian models, we have developed a high-throughput screen assay to test reproductive toxicity in the roundworm C. elegans. We have mobilized several important features offered by this commonly used genetic model system such as its small size, low cost, short reproduction cycle, high proportion of germ cells and ease of manipulation6. Worms can be grown in 96-well plates or in high volume liquid cultures and because of their transparency can be directly imaged on plates for detection of fluorescent reporters. The assay described below takes advantage of these characteristics and takes advantage of a worm strain containing the fluorescent reporter Pxol-1::GFP to detect germline disruption and induction of embryonic aneuploidy.
The use of this reporter strain is based on the generally rare occurrence of males in a mainly hermaphroditic worm population. These males (&#60;0.2%) naturally originate from error in the segregation of the X chromosome7. However, as germline disruption frequently leads to errors in segregation of autosomes and of heterochromosomes, it correlates both with an elevated incidence of males phenotype (X missegregation) as well as embryonic lethality (autosome missegregation). To easily detect the induction of males while circumventing the issue of embryonic lethality, a male-specific promoter (xol-1) is used to drive expression of GFP in early embryos still contained within the worm&#8217;s uterus. As such, the appearance of GFP-expressing embryos is used as a proxy for the presence of aneuploid embryos. This method has been previously used to identify genes implicated in germline maintenance and meiosis8,9. Adapted to chemical screening, this strain is employed in a medium to high-throughput screen. Importantly, the strain faithfully reports the aneugenicity of chemicals and is therefore relevant to mammalian reproductive endpoints10. The assay described here will be particularly useful to toxicologists in pharmaceutical and chemical industry settings looking to rapidly assess the toxicity of chemicals towards reproductive endpoints. Furthermore, this assay fully aligns with the governmental priorities highlighted in the Toxicity in the 21st century report11.

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			<td height="21" style="height:21px;width:473px;">Name of Reagent/ Equipment</td>
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			<td style="width:119px;">Catalog Number</td>
			<td style="width:255px;">Comments/Description</td>
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			<td height="21" style="height:21px;">60mm vented, sharp edge Petri Dishes</td>
			<td style="width:129px;">Tritech</td>
			<td>T3315</td>
			<td style="width:255px;"></td>
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			<td height="21" style="height:21px;width:473px;">Agar</td>
			<td>Apex</td>
			<td>20-274</td>
			<td style="width:255px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:473px;">Axygen 96 Well Clear Round Bottom 2mL Polypropylene Deep Well Plate</td>
			<td style="width:129px;">Corning</td>
			<td>P-DW-20-C-S</td>
			<td style="width:255px;"></td>
		</tr>
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			<td height="21" style="height:21px;width:473px;">Bactopeptone</td>
			<td style="width:129px;">Apex</td>
			<td>20-261</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:473px;">Bacto-tryptone</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP1421-2</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:473px;">Bleach</td>
			<td style="width:129px;">Clorox</td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
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			<td height="21" style="height:21px;">Calcium Chloride (CaCl2)</td>
			<td style="width:129px;">VWR</td>
			<td>AA12316-A1</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cholesterol</td>
			<td>Fisher Scientific</td>
			<td>ICN10138225</td>
			<td style="width:255px;"></td>
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			<td height="21" style="height:21px;width:473px;">DMSO</td>
			<td style="width:129px;">VWR</td>
			<td>IC19018680</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">E. coli (OP50)</td>
			<td style="width:129px;">Caenorhabditis Genetics Center</td>
			<td style="width:119px;"></td>
			<td>http://www.cbs.umn.edu/cgc</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:473px;">Ethanol 200 proof</td>
			<td style="width:129px;">VWR</td>
			<td>EM-4455S</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Greiner CELLSTAR 384 well plates</td>
			<td>Sigma-Aldrich</td>
			<td>M1937-32EA</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageXpress Micro XLS System&#160;</td>
			<td style="width:129px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
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			<td height="21" style="height:21px;width:473px;">Levamisole hydrochloride&#160;</td>
			<td style="width:129px;">Fluka</td>
			<td style="width:119px;">31742</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Sulfate Anhydrous (MgSO4)</td>
			<td style="width:129px;">VWR</td>
			<td>97061-438</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MetaXpress High Content Image Acquisition &#38; Analysis Software&#160;</td>
			<td style="width:129px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
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			<td height="21" style="height:21px;width:473px;">Potassium phosphate monobasic (KH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td style="width:255px;"></td>
		</tr>
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			<td height="21" style="height:21px;width:473px;">Rayon Films for Biological Cultures</td>
			<td style="width:129px;">VWR</td>
			<td>60941-086</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:473px;">Sodium hydroxide (NaOH)</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S318</td>
			<td style="width:255px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:473px;">Sodium Phosphate Dibasic (Na2HPO4)</td>
			<td style="width:129px;">VWR</td>
			<td>BDH0316</td>
			<td style="width:255px;"></td>
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			<td height="84" style="height:84px;width:473px;">Stereomicroscope&#160;</td>
			<td style="width:129px;">Nikon</td>
			<td style="width:119px;">SMZ 745</td>
			<td style="width:255px;">This microscope has a total magnification form 3.35x to 300x. Any microscope with similar characteristics will work.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:473px;">TY2441 C. elegans, Pxol-1::GFP reporter strain&#160;</td>
			<td style="width:129px;">Caenorhabditis Genetics Center</td>
			<td style="width:119px;"></td>
			<td>http://www.cbs.umn.edu/cgc</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:473px;">Yeast extract</td>
			<td style="width:129px;">Becton Dickinson</td>
			<td style="width:119px;">212750</td>
			<td style="width:255px;"></td>
		</tr>
	</tbody>
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comprehensive assessment of germline chemical toxicity using the nematode caenorhabditis elegans

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.

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 ...

Watch this Scientific Journal Video about Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans at JoVE.com

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

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.

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

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