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 tubes. Pellet the bacteria by centrifuging for 5 min at 6,000 rpm (5,000 x g).</li>
	<li>Discard the supernatant and wash the bacteria with 50 ml of sterile M9. Repeat twice.</li>
	<li>After the second wash remove the remaining M9, ensuring that no M9 is left in the tube.</li>
	<li>Determine the weight of the pellet by weighing the tube containing the bacteria pellet and subtract the weight of the 50 ml conical tube.</li>
	<li>Resuspend the pellet in M9 at a concentration of 100 mg/ml.</li>
	<li>Store the OP50 solution at 4 &#176;C until it is used for the worm culture.</li>
</ol>
2. Preparation of Nematode Growth Medium (NGM) Petri Plates
NOTE: This section describes the preparation of NGM Petri plates, which are the plates where the C. elegans worms are routinely maintained in the laboratory.
<ol>
	<li>Autoclave the NGM agar medium.</li>
	<li>Using sterile procedures, dispense 17 ml of NGM agar solution into 6 cm Petri plates using a peristaltic pump. 
	NOTE: A constant amount of agar in the plates reduces the need for refocusing the microscope when switching from one plate to another.</li>
	<li>Leave the plates at room temperature for 2-3 days before use to let the agar solidify and allow the excess moisture to evaporate.</li>
	<li>Seed the NGM plates using a sterile technique. Apply 1-2 drops of OP50 liquid culture and spread using a glass rod. Make sure not to spread the bacteria lawn all the way to the edges of the plates.</li>
	<li>Allow the OP50 lawn to grow for 1-2 days at room temperature before using the plates to grow worms.</li>
</ol>
3. Preparation of a Synchronous Worm Population
NOTE: The life cycle of C. elegans is comprised of the embryonic stage, four larval stages (L1-L4) and adulthood. This section describes the preparation of an age-synchronous population of worms. All materials coming in contact with C.elegans after the bleach treatment must be sterile.
<ol>
	<li>Day 1 &#8211; Preparation of Gravid Worm Population
	<ol>
		<li>Use NGM plates in which the majority of the worm population consists of starved L1 larvae.</li>
		<li>Collect the L1 larvae with a sterilized worm pick and transfer them to fresh 6 cm NGM plates seeded with OP50. Incubate for approximately 65 hr at 20 &#176;C until the majority of the worms are gravid adults. To avoid plate overcrowding and allow the worms to grow without depleting the bacteria, do not transfer more than one or two clusters of L1 larvae to each fresh NGM plate.</li>
	</ol>
	</li>
	<li>Day 4 &#8211; Establishment of a Synchronized L1 Larvae Population
	<ol>
		<li>Collect the gravid worms from the NGM plates by washing them with 1-2 ml of M9 and transfer them to a conical 15 ml tube.</li>
		<li>Let the gravid worms sediment to the bottom of the 15 ml conical tube for approximately 5-10 min. Remove the supernatant without disturbing the worm pellet.</li>
		<li>Transfer the worm pellet to 2-4 microcentrifuge tubes and add 1 ml of bleach/NaOH solution. Incubate for 2-3 min at RT. Monitor the progress of the reaction under the stereomicroscope to confirm all the worms are dead. Do not bleach longer than 5 min as the embryos could die.</li>
		<li>Collect the worms by centrifuging for 1 min at 3,000 rpm (900 x g). Remove the supernatant and add 1 ml of sterile M9 to neutralize the reaction.</li>
		<li>Wash the worms twice by centrifuging for 1 min at 3,000 rpm (900 x g).</li>
		<li>Remove the supernatant and add M9 to up to 100-200 &#181;l.</li>
		<li>Using a glass Pasteur pipette add a drop of the worm/M9 mix to clear NGM plates and incubate them for at least 24 hr at 20 &#176;C to allow the embryos to hatch. 
		NOTE: Without food the larvae&#8217;s growth will be halted at the L1 stage.</li>
	</ol>
	</li>
	<li>Day 5 &#8211; Establishment of a Synchronized L4 Larvae Population
	<ol>
		<li>Collect the L1 larvae from the clear NGM plates by washing the plates with 1-2 ml of M9 and transfer them to a conical 15 ml tube.</li>
		<li>Let the dead adult worms sediment to the bottom of the 15 ml conical tube for approximately 5 min.</li>
		<li>Collect the supernatant, where the L1 worms are, in a new conical 15 ml tube, and precipitate the worms by centrifuging for 2 min at 2,600 rpm (1,000 x g).</li>
		<li>Remove the supernatant leaving approximately 500 &#181;l.</li>
		<li>Determine the concentration of worms in the M9 solution by counting the number of worms in 10 &#181;l drops using a stereomicroscope. Count at least 5 drops.</li>
		<li>Adjust the concentration of the worms to 20-25 worms/&#181;l and add 50 &#181;l of the worm/M9 mix to NGM plates seeded with OP50.</li>
		<li>Incubate for 65 hr at 15 &#176;C (for incubation through the week-end) to allow the L1 synchronized population to grow until they reach the L4 stage.</li>
	</ol>
	</li>
</ol>
4. Exposure of Worms to Chemicals in 96-well Plates
NOTE: This section describes the use of Pxol1::gfp transcriptional reporter containing C. elegans strain to screen for the induction of aneuploidy.
<ol>
	<li>Collect the L4 larvae from the clear NGM plates by washing the plates with 1-2 ml of M9 and transfer them to a conical 15 ml tube.</li>
	<li>Let the L4 worms sediment to the bottom of the 15 ml conical tube for approximately 5-10 min. Remove the supernatant without disturbing the worm pellet.</li>
	<li>Add 3-5 ml of M9 and determine the concentration of worms in the M9 solution by counting the number of worms in 10 &#181;l drops using a stereomicroscope. Count at least 5 drops for each sample.</li>
	<li>Resuspend the worms at a concentration of 1,000 worms/ml in M9.</li>
	<li>Dilute the OP50 bacteria from section 1 10-fold with M9. Make sure the resuspended bacteria reach room temperature because lower temperature affects worm development.</li>
	<li>Using a multichannel pipette, first add 100 &#181;l of the worm/M9 mix (from step 4.4) and then add 400 &#181;l of the diluted OP50 bacteria (from step 4.5) to each well of a 2 ml deep round bottom 96-well plate. At the end, each well will have 100 worms in 500 &#181;l.</li>
	<li>Add 0.5 &#181;l of either the test chemical or the appropriate control to the desired wells, to achieve a suggested final concentration of 100 &#181;M, a concentration commonly used in chemical screens in C. elegans12. Expose ethanol or DMSO solvent controls at a final concentration of 0.1%. 
	NOTE: We recommend the use of nocodazole (100 &#181;M) as positive control.</li>
	<li>Seal the plate using adhesive film. Be sure to seal the plate well to prevent cross contamination between wells.</li>
	<li>Wrap the plate with aluminum foil.</li>
	<li>Transfer the plate to a shaker (170-180 rpm) of the appropriate temperature for the suitable length of time (24 or 65 hr). The temperature of the room affects the growth of the worms; maintain the temperature around 20 &#176;C.</li>
</ol>
5. Image Acquisition of Gravid Worms in a 384-well Plate
NOTE: This section describes the use of a high content microscope to image the exposed Pxol1::gfp worms, to visualize the expression of GFP in the embryos within the uterus of adult hermaphrodites. For each 384-well plate to be screened, use worms from 4 x 96-well plates.
<ol>
	<li>After chemical exposure in the shaker, let the 96-well plate rest for 10-15 min to allow the adult worms to sediment to the bottom of the plate.</li>
	<li>Using a multichannel pipette, remove 350 &#181;l of the M9, being very careful not to disturb the worms in the bottom of the plate.</li>
	<li>Wash the worms with 1 ml of M9 and repeat step 5.1.</li>
	<li>Using a multichannel pipette, remove 1 ml of the M9, being vey careful not to disturb the worms in the bottom of the plate.</li>
	<li>Using a multichannel pipette, resuspend the worms in the remaining M9 and collect 100 &#181;l of the worm/M9 mix from the 96-well plates and load it into the wells of a black walls/clear bottom 384-well plate. Repeat the process using worms from 4 x 96-well plates, until all the wells of the 384-well plate have been loaded. 
	NOTE: It is important for all the wells to have the same volume in order to increase the efficiency and speed of the autofocus during the image acquisition process. Each well should contain between 80 to 100 worms.</li>
	<li>Add 1 &#181;l of levamisole (100 &#181;M) to each well and let the worms incubate for approximately 30 min. 
	NOTE: Levamisole acts as an acetylcholine receptor agonist and immobilizes the worms.</li>
	<li>Transfer the plate to a widefield high content microscope capable of providing automated imaging.</li>
	<li>For image acquisition, use a high content image acquisition and analysis software according to the manufacturer&#8217;s guidelines. Select the 4X objective to acquire 1 image per well.</li>
	<li>Before starting the image acquisition, adjust the GFP imaging parameters to 45 msec of exposure and image resolution to 2,160 x 2,160.</li>
	<li>Collect the data as the number of GFP positive worms divided by the total number of worms in the well, the latter measure being relatively consistent between wells.</li>
</ol>

<ol>
	<li>Office, U. G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Report+No.+GAO-06-1032T.">Report No. GAO-06-1032T.</a> Actions are needed to improve the effectiveness of EPA&#8217;s chemical review program. Testimony before the Committee on Environment and Public Works. US Senate. , (2009).</li><li>Fragouli, E., Wells, D., Delhanty, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+abnormalities+in+the+human+oocyte.">Chromosome abnormalities in the human oocyte.</a> Cytogenet Genome Res. 133 (2-4), 107-118 (2011).</li><li>Hassold, T., Hunt, P. A., Sherman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trisomy+in+humans:+incidence,+origin+and+etiology.">Trisomy in humans: incidence, origin and etiology.</a> Curr Opin Genet Dev. 3 (3), 398-403 (1993).</li><li>Heron, M., Hoyert, D. L., Murphy, S. L., Xu, J., Kochanek, K. D., Tejada-Vera, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deaths:+final+data+for+2006.">Deaths: final data for 2006.</a> Natl Vital Stat Rep. 57 (14), 1-134 (2009).</li><li>Hunt, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meiosis+in+mammals:+recombination,+non-disjunction+and+the+environment.">Meiosis in mammals: recombination, non-disjunction and the environment.</a> Biochem Soc Trans. 34 (Pt 4), 574-577 (2006).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77 (1), 71-94 (1974).</li><li>Hodgkin, J., Horvitz, H. R., Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nondisjunction Mutants of the Nematode CAENORHABDITIS ELEGANS.Genetics. 91 (1), 67-94 (1979).</li><li>Nicoll, M., Akerib, C. C., Meyer, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-chromosome-counting+mechanisms+that+determine+nematode+sex.">X-chromosome-counting mechanisms that determine nematode sex.</a> Nature. 388 (6638), 200-204 (1997).</li><li>Kelly, K. O., Dernburg, A. F., Stanfield, G. M., Villeneuve, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+msh-5+is+required+for+both+normal+and+radiation-induced+meiotic+crossing+over+but+not+for+completion+of+meiosis.">Caenorhabditis elegans msh-5 is required for both normal and radiation-induced meiotic crossing over but not for completion of meiosis.</a> Genetics. 156 (2), 617-630 (2000).</li><li>Allard, P., Kleinstreuer, N. C., Knudsen, T. B., Colaiacovo, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Screening+Platform+for+the+Rapid+Assessment+of+Chemical+Disruption+of+Germline+Function.">A Screening Platform for the Rapid Assessment of Chemical Disruption of Germline Function.</a> Environ Health Perspect. 121 (6), 717-724 (2013).</li><li>National Research Council. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Toxicity Testing in the 21st Century: A Vision and a Strategy. , (2007).</li><li>Boyd, W. A., Smith, M. V., Kissling, G. E., Freedman, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medium-+and+high-throughput+screening+of+neurotoxicants+using+C.+elegans.">Medium- and high-throughput screening of neurotoxicants using C. elegans.</a> Neurotoxicol Teratol. 32 (1), 68-73 (2010).</li></ol>

The authors declare that they have no competing financial interests.

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.

<table border="0" cellpadding="0" cellspacing="0" width="975">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:473px;">Name of Reagent/ Equipment</td>
			<td style="width:129px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:255px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:473px;">Agar</td>
			<td>Apex</td>
			<td>20-274</td>
			<td style="width:255px;"></td>
		</tr>
		<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>
		<tr height="21">
			<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>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		<tr height="21">
			<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>
		<tr height="21">
			<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>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="84">
			<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>
</table>

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 ...

comprehensive-assessment-germline-chemical-toxicity-using-nematode

Research

JoVE Journal

Developmental Biology

8.0K Views.  University of California, Los Angeles. 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.

Video: Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically.

The protocols describe two in vitro developmental toxicity test systems (UKK and UKN1) based on human embryonic stem cells and transcriptome studies. The test systems predict human developmental toxicity hazard, and may contribute to reduce animal studies, costs and the time required for chemical safety testing.

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in ...

Salinity-dependent Toxicity Assay of Silver Nanocolloids Using Medaka Eggs

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and seawater. Tests of the toxicity of chemicals and assessments of their ecological risks to aquatic organisms are frequently performed in freshwater, but the toxicity of chemicals to aquatic organisms depends on pH, temperature, and salinity. There is no method, however, for testing the salinity dependence of toxicity to aquatic organisms. Here, we used medaka (Oryzias latipes) because they can adapt to freshwater, brackish water, and seawater. Different concentrations of embryo-rearing medium (ERM) (1x, 5x, 10x, 15x, 20x, and 30x) were employed to test the toxicity of silver nanocolloidal particles (SNCs) to medaka eggs (1x ERM and 30x ERM have osmotic pressures equivalent to freshwater and seawater, respectively). In six-well plastic plates, 15 medaka eggs in triplicate were exposed to SNCs at 10 mg/L&#8722;1 in different concentrations of ERM at pH 7 and 25 &#176;C in the dark.
We used a dissecting microscope and a micrometer to measure heart rate per 15 sec and eye diameter on day 6 and full body length of the larvae on hatching day (section 4). The embryos were observed until hatching or day 14; we then counted the hatching rate every day for 14 days (section 4). To see silver accumulation in embryos, we used inductively coupled plasma mass spectrometry to measure the silver concentration of test solutions (section 5) and dechorionated embryos (section 6).The toxicity of the SNCs to medaka embryos obviously increased with increasing salinity. This new method allows us to test the toxicity of chemicals in different salinities.

Embryonic stages are the most susceptible to xenobiotics. Although chemical toxicity depends on salinity, no method exists to test the salinity dependence of toxicity to aquatic organisms. Here, we describe a new and high-throughput method for determining the salinity dependence of toxicity to aquatic embryos.

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Caenorhabditis elegans (C. elegans) is a good model to study axonal and intracellular transport. Here, I describe a protocol for in vivo recording and analysis of axonal and intraflagellar transport in C. elegans.

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are facilitated by the transparency of the animal, the ability to do forward and reverse genetic assays, the relative ease of generating fluorescently labeled proteins, and the use of fluorescent dyes that can either be microinjected into the early embryo or incorporated into its food (E. coli strain OP50) to label cellular organelles (e.g. 9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1&#39;H-2,2&#39;-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron). Here, we present the use of a fluorescent pH-sensitive dye that stains intestinal lysosomes, providing a visual readout of dynamic, physiological changes in lysosomal acidity in live worms. This protocol does not measure lysosomal pH, but rather aims to establish a reliable method of assessing physiological relevant variations in lysosomal acidity. cDCFDA is a cell-permeant compound that is converted to the fluorescent fluorophore 5-(and-6)-carboxy-2&#39;,7&#39;-dichlorofluorescein (cDCF) upon hydrolysis by intracellular esterases. Protonation inside lysosomes traps cDCF in these organelles, where it accumulates. Due to its low pKa of 4.8, this dye has been used as a pH sensor in yeast. Here we describe the use of cDCFDA as a food supplement to assess the acidity of intestinal lysosomes in C. elegans. This technique allows for the detection of alkalinizing lysosomes in live animals, and has a broad range of experimental applications including studies on aging, autophagy, and lysosomal biogenesis.

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

A step-by-step guide to probe loss of lysosomal acidity in the intestine of C. elegans using the pH-sensitive vital dye 5(6)-carboxy-2&#39;,7&#39;-dichlorofluorescein diacetate (cDCFDA)

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases of the cell cycle, or the incorporation of nucleoside analogs. During S-phase, DNA polymerases incorporate thymidine analogs such as EdU or BrdU into chromosomal DNA, marking the cells for analysis. For C. elegans, the nucleoside analog EdU is fed to the worms during regular culture and is compatible with immunofluorescent techniques. The germline of C. elegans is a powerful model system for the studies of signaling pathways, stem cells, meiosis, and cell cycle because it is transparent, genetically facile, and meiotic prophase and cellular differentiation/gametogenesis occur in a linear assembly-like fashion. These features make EdU a great tool to study dynamic aspects of mitotically cycling cells and germline development. This protocol describes how to successfully prepare EdU bacteria, feed them to wild-type C. elegans hermaphrodites, dissect the hermaphrodite gonad, stain for EdU incorporation into DNA, stain with antibodies to detect various cell cycle and developmental markers, image the gonad and analyze the results. The protocol describes the variations in the method and analysis for the measurement of S-phase index, M-phase index, G2 duration, cell cycle duration, rate of meiotic entry, and rate of meiotic prophase progression. This method can be adapted to study the cell cycle or cell history in other tissues, stages, genetic backgrounds, and physiological conditions.

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

An imaging-based method is described that can be used to identify S-phase and analyze cell cycle dynamics in the C. elegans hermaphrodite germline using the thymidine analog EdU. This method requires no transgenes and is compatible with immunofluorescent staining.

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases ...

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented protocol uses this model to determine the pathogenicity caused by the mitis group streptococci via the production of H2O2. The mitis group streptococci are an emerging threat that cause many human diseases such as bacteremia, endocarditis, and orbital cellulitis. Described here is a protocol to determine the survival of these worms in response to H2O2 produced by this group of pathogens. Using the gene skn-1 encoding for an oxidative stress response transcription factor, it is shown that this model is important for identifying host genes that are essential against streptococcal infection. Furthermore, it is shown that activation of the oxidative stress response can be monitored in the presence of these pathogens using a transgenic reporter worm strain, in which SKN-1 is fused to green fluorescent protein (GFP). These assays provide the opportunity to study the oxidative stress response to H2O2 derived by a biological source as opposed to exogenously added reactive oxygen species (ROS) sources.

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

The nematode Caenorhabditis elegans is an excellent model to dissect host-pathogen interactions. Described here is a protocol to infect the worm with members of the mitis group streptococci and determine activation of the oxidative stress response against H2O2 produced by this group of organisms.

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented ...

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female reproductive tract. While in vitro studies suggest that sperm of internally fertilizing animals can respond to various cues from their surroundings, the inability to visualize their behavior inside the female reproductive tract creates a challenge for understanding sperm migration and mobility in its native environment. Here, we describe a method using C. elegans that overcomes this limitation and takes advantage of their transparent epidermis. C. elegans males stained with a mitochondrial&#160; dye are mated with adult hermaphrodites, which act as modified females, and deposit fluorescently labeled sperm into the hermaphrodite uterus. The migration and motility of the labeled sperm can then be directly tracked using an epi-fluorescence microscope in a live hermaphrodite. In wild-type animals, approximately 90% of the labeled sperm crawl through the uterus and reach the fertilization site, or spermatheca. Images of the uterus can be taken 1 h after mating to assess the distribution of the sperm within the uterus and the percentage of sperm that have reached the spermatheca. Alternatively, time-lapse images can be taken immediately after mating to assess sperm speed, directional velocity and reversal frequency. This method can be combined with other genetic and molecular tools available for the C.elegans to identify novel genetic and molecular mechanisms that are important in regulating sperm guidance and motility within the female reproductive tract.

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Sperm must successfully navigate through the oviduct to fertilize an oocyte. Here, we describe an assay for measuring sperm migration within the C. elegans hermaphrodite uterus. This assay can provide quantitative data on sperm distribution within the uterus after mating, as well as on speed, directional velocity, and reversal frequency.

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female ...

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological conditions. Post-mitotic neurons represent a major cell type affected by aging. Although multiple mammalian models of neuronal aging exist, they are challenging and expensive to establish. The roundworm Caenorhabditis elegans is a powerful model to study neuronal aging, as these animals have short lifespan, an available robust genetic toolbox, and well-cataloged nervous system. The method presented herein allows for seamless isolation of specific cells based on the expression of a transgenic green fluorescent protein (GFP). Transgenic animal lines expressing GFP under distinct, cell type-specific promoters are digested to remove the outer cuticle and gently mechanically disrupted to produce slurry containing various cell types. The cells of interest are then separated from non-target cells through fluorescence-activated cell sorting, or by anti-GFP-coupled magnetic beads. The isolated cells can then be cultured for a limited time or immediately used for cell-specific ex vivo analyses such as transcriptional analysis by real time quantitative PCR. Thus, this protocol allows for rapid and robust analysis of cell-specific responses within different neuronal populations in C. elegans.

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

Here, we present a protocol for simple isolation of specific groups of live neuronal cells expressing green fluorescent protein from transgenic Caenorhabditis elegans lines. This method enables a variety of ex vivo studies focused on specific neurons and has the capacity to isolate cells for further short-term culturing.

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological ...