Support by NSF (EPSCoR EPS-0814251) and NIH (1P20GM103653-01A1) to MKT and HSD, Delaware State University&#39;s institutional student support to MDM (Title III HBGI) and KRS (NIH-INBRE) is gratefully acknowledged. Thanks to the C. elegans Genetic Center (supported by NIH Office of Research Infrastructure Programs P40 OD010110) for C. elegans wild-type N2 strain.

Protocols given below have been standardized to test the effects of BPA exposure during early embryogenesis, and may be modified for use with BPS, BPF or other EDCs and toxicants on C. elegans embryos.
1. Material Setup
<ol>
	<li>Prepare 500 mL of Nematode Growth Media (NGM)18 media by dissolving 1.5 g of NaCl, 1.25 g of peptone, and 8.5 g of agar in water to 500 mL. After autoclaving (121 &#176;C, 15 PSI, 20 min), add 0.5 mL of cholesterol (5 mg/mL in ethanol), 0.5 mL of 1 M MgSO4, 0.5 mL of 1 M CaCl2 and 12.5 mL of 1 M potassium phosphate buffer, pH 7.4 (108.3 g of KH2PO4, 35.6 g of K2HPO4, water to 1 L).</li>
	<li>Prepare hypochlorite solution by mixing 25 mL of 1 N KOH, 4 mL of bleach, and 71 mL of water. Make this solution fresh.</li>
	<li>Prepare M9 Buffer by mixing 3 g of KH2PO4, 6 g of Na2HPO4, 5 g of NaCl, 5 mL of 1 M of MgSO4, and water to 1 L.</li>
	<li>Prepare S Buffer18 by mixing 129 mL of 0.02 M K2HPO4, 871 mL of KH2PO4, and 5.85 g of NaCl.</li>
	<li>Sterilize M9 and S buffers by autoclaving.</li>
	<li>Grow an overnight culture of Escherichia coli (OP 50 strain) in 20 mL of Luria Broth (LB).</li>
	<li>Dissolve BPA in 10% ethanol to make a 1 mM stock solution. Make subsequent dilutions of 0.1 &#181;M, 0.5 &#181;M, 1 &#181;M, 5 &#181;M and 10 &#181;M in S buffer. 
	NOTE: Dilution in the aqueous S buffer greatly reduces the concentration of ethanol; for example, at the highest BPA concentration described in this protocol (10 &#181;M), the concentration of ethanol is 0.1% v/v.</li>
</ol>
2. C. elegans Culturing and Synchronization
<ol>
	<li>Pour the NGM media into 100 mm plates (about 25 mL/plate) and allow it to solidify. Once solidified, seed the plates by spreading 150 &#181;L of E. coli OP50 from the liquid culture grown overnight. Incubate plates at 37 &#176;C overnight.</li>
	<li>The next day, transfer N2 worms onto the new plates by chunking an approximate slab of 1 cm square. Allow worms to grow for 3 days at 20 &#176;C until the plate is populated with well-fed mature adults containing visible embryos.</li>
	<li>To synchronize, wash each plate by pipetting up to 5 mL of M9 buffer across the plate. Transfer the liquid to a sterile 15 mL conical centrifuge tube. 
	NOTE: Synchronization is done to kill adult worms and collect embryos that are relatively the same stage.</li>
	<li>Centrifuge at 3,000 x g for 4 min at room temperature in order to obtain a loose pellet of adult worms.</li>
	<li>Using a 10 mL pipette, carefully remove the supernatant so as to leave the pellet intact. Add 5 mL of the hypochlorite solution and gently mix.</li>
	<li>Centrifuge at 3,000 x g at room temperature for 4 minutes.</li>
	<li>Remove supernatant with a 10 mL pipette and wash with 7 mL of M9 buffer. Repeat this step twice.</li>
</ol>
3. Exposing Worms to BPA
<ol>
	<li>After the last wash from step 2.7 above, suspend about 100 &#181;L of eggs with M9 buffer. Transfer about 50 &#181;L of eggs to 2 mL microfuge tubes already containing the appropriate concentration BPA diluted in S buffer. Adjust the amount of S-buffer to ensure the correct final concentration of BPA (0.0 &#181;M, 0.1 &#181;M, 0.5 &#181;M, 1.0 &#181;M, 5.0 &#181;M and 10 &#181;M) per tube. Table 1 illustrates one way to make these dilutions.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>For Final BPA Concentration</td>
			<td>Stock BPA (100 &#956;M)</td>
			<td>S Buffer</td>
			<td>Synchronized Worms</td>
		</tr>
		<tr>
			<td>0.1 &#956;M</td>
			<td>1 &#956;L</td>
			<td>949 &#956;L</td>
			<td>50 &#956;L</td>
		</tr>
		<tr>
			<td>0.5 &#956;M</td>
			<td>5 &#956;L</td>
			<td>945 &#956;L</td>
			<td>50 &#956;L</td>
		</tr>
		<tr>
			<td>1 &#956;M</td>
			<td>10 &#956;L</td>
			<td>940 &#956;L</td>
			<td>50 &#956;L</td>
		</tr>
		<tr>
			<td>5 &#956;M</td>
			<td>50 &#956;L</td>
			<td>900 &#956;L</td>
			<td>50 &#956;L</td>
		</tr>
		<tr>
			<td>10 &#956;M</td>
			<td>100 &#956;L</td>
			<td>850 &#956;L</td>
			<td>50 &#956;L</td>
		</tr>
	</tbody>
</table>
Table 1: Stock BPA, S buffer and synchronized worms used to attain the desired concentrations used for our study.
<ol start="2">
	<li>Place tubes on a shaker and gently shake for 4 h at 20&#176;C, at 25 rpm for a rotary shaker or 25 tilts/min for a rocking shaker.</li>
	<li>After 4 h, place tubes in tube holders and allow worms to settle.</li>
	<li>Discard supernatant and transfer worms to seeded plates (NGM plates with OP50 E. coli). Make the liquid OP50 E. coli culture as stated in 1.5 and seed the plates as stated in 2.1.</li>
	<li>Let the worms grow for 60 h in 20 &#176;C. Examine plates every day for contamination. Contaminated NGM plates are usually discolored and accompanied by individual colonies. Check worms under the microscope for overcrowding. A lack of OP50 E. coli lawn and concentrated worms in small spots signify overcrowding.</li>
</ol>
4. Anterior Touch Habituation Assay
<ol>
	<li>Transfer approximately 10 synchronized young adult worms to new unseeded NGM plates using a fire-sterilized 30 G platinum wire pick. Leave worms undisturbed for 5 min to allow them time to acclimatize to the new plate.</li>
	<li>For the habituation response, use an eyebrow hair attached to the end of a wooden skewer or toothpick, and sterilize by dipping in 70% ethanol. Wipe with a clean lint-free tissue and wait for 1 min for ethanol to evaporate off. Gently touch the worm on the head (anterior of the pharyngeal bulb) using the eyebrow hair. Repeat the touches, allowing 10 s in between touches in order to allow the worm to recover.</li>
	<li>Continue to touch (allowing 10 s interstimulus intervals) until the worm no longer moves backwards. At this point, the worm has habituated to the stimulus. Record the number of touches required for the worm to habituate.</li>
	<li>Analyze the difference between the mean habituation rates of the treated and untreated controls using one-way ANOVA followed by Tukey&#39;s post hoc test.</li>
</ol>
5. Worm Fecundity Assay
<ol>
	<li>Transfer individual L3 worms to a fresh and seeded plate using a platinum pick. Make sure that only one worm is placed per plate. 
	NOTE: After the L3 larval stage, worms develop into L4 larval stage and adulthood. Picking them early is convenient in that they are large enough for the transfer of individual animals while ensuring that they have not already laid any eggs which otherwise would be missed in the count.</li>
	<li>Count the numbers of eggs laid every 12&#8211;24 h. After counting, transfer the parent worm to a new plate. Repeat until the worm stops laying eggs, typically after 5 days when incubated at 20 &#176;C. 
	NOTE: On average, a wild type worm can lay up to 300 eggs; therefore, transferring the parent to a new plate daily prevents overcrowding.</li>
	<li>Analyze the difference between the average number of eggs laid by the treated and untreated controls using one-way ANOVA followed by Tukey&#39;s post hoc test.</li>
</ol>

<ol>
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Endocrinol. 13, 161-173 (2017).</li><li>Melnik, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Summary+of+the+National+Toxicology+Program's+Report+of+the+Endocrine+Disruptors+Low-Dose+Peer+Review.">Summary of the National Toxicology Program's Report of the Endocrine Disruptors Low-Dose Peer Review.</a> Environ. Health Perspect. 110, 427-431 (2002).</li><li>Kortenkamp, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ten+years+of+mixing+cocktails:+a+review+of+combination+effects+of+endocrine-disrupting+chemicals.">Ten years of mixing cocktails: a review of combination effects of endocrine-disrupting chemicals.</a> Environ. Health Perspect. 115 Suppl 1, 98-105 (2007).</li><li>Vandenberg, L. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormones+and+endocrine-disrupting+chemicals:+low-dose+effects+and+nonmonotonic+dose+responses.">Hormones and endocrine-disrupting chemicals: low-dose effects and nonmonotonic dose responses.</a> Endocr. Rev. 33, 378-455 (2012).</li><li>Eramo, S., Urbani, G., Sfasciotti, G. L., Brugnoletti, O., Bossu, M., Polimeni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogenicity+of+bisphenol+A+released+from+sealants+and+composites:+a+review+of+the+literature.">Estrogenicity of bisphenol A released from sealants and composites: a review of the literature.</a> Ann Stomatol. (Roma). 1, 14-21 (2010).</li><li>Cantonwine, D. E., Ferguson, K. K., Mukherjee, B., McElrath, T. F., Meeker, J. 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Disord. 16, 289-297 (2015).</li><li>Mimoto, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+an+estrogenic+hormone+receptor+in+Caenorhabditis+elegans.">Identification of an estrogenic hormone receptor in Caenorhabditis elegans.</a> Biochem. Biophys. Res. Commun. 364, 883-888 (2007).</li><li>Allard, P., Colaiacovo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bisphenol+A+impairs+the+double-strand+break+repair+machinery+in+the+germline+and+causes+chromosome+abnormalities.">Bisphenol A impairs the double-strand break repair machinery in the germline and causes chromosome abnormalities.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 20405-20410 (2010).</li><li>Mersha, M. D., Patel, B. M., Patel, D., Richardson, B. N., Dhillon, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+BPA+and+BPS+exposure+limited+to+early+embryogenesis+persist+to+impair+non-associative+learning+in+adults.">Effects of BPA and BPS exposure limited to early embryogenesis persist to impair non-associative learning in adults.</a> Behav. Brain. Funct. 11, 27 (2015).</li><li>Chen, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+to+the+BPA-Substitute+Bisphenol+S+Causes+Unique+Alterations+of+Germline+Function.">Exposure to the BPA-Substitute Bisphenol S Causes Unique Alterations of Germline Function.</a> PLoS Genet. 12, e1006223 (2016).</li><li>Rankin, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Habituation+revisited:+an+updated+and+revised+description+of+the+behavioral+characteristics+of+habituation.">Habituation revisited: an updated and revised description of the behavioral characteristics of habituation.</a> Neurobiol. Learn. Mem. 92, 135-138 (2009).</li><li>Hope, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> C elegans: A Practical Approach. , (1999).</li><li>Sanchez, K., Mersha, M., Dhillon, H. 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The authors declare that they have nothing to disclose.

Phases of rapid growth and development such as embryogenesis are particularly vulnerable to the detrimental effects of various endocrine-disrupting compounds, including BPA. We provide detailed protocols for studying the effects of exposure to BPA or other toxicants in the C. elegans invertebrate model, which allows convenient titration of a range of concentrations to assess its effect on embryo viability (Figure 2). Follow up behavioral experiments on surviving adults that develop from embryos that were exposed to low doses of BPA allow assaying long-term effects on neuronal function (Figure 3). The central idea of this paper is to provide detailed protocols for exposing embryos to various toxicants during the early embryogenesis period in the C. elegans model; the first 4 h window of its 11.5 hour embryonic development period (at 20 &#176;C) corresponds to the proliferation period, which is followed by organogenesis. Towards this end, we have provided validation of the end points through fecundity and non-associative learning assays. While the mechanistic basis of the action of BPA and other bisphenols is the holy grail towards which research in this field is aimed, we have not attempt to offer a mechanistic explanation for the action of BPA in this methodology focused article. However, we would like to point out that the C. elegans model offers an ideal system to further dissect the mechanism of action. For example, in future work on deciphering mechanisms of BPA action, C. elegans mutants can be generated and screened for a resistance to the effects of BPA and thus pave the way for deciphering the pathway. Additionally, our companion paper deals with the effect of BPA at the neural network synchrony level in vertebrates, providing another avenue to understand the basis for investigating potential mechanisms for toxicant effects19.
In the methodology detailed in this paper, some of the critical steps that should be executed extra carefully include the following: Choosing a plate with well-fed C. elegans adults with mature eggs visible under the dissection microscope is crucial, and failing to follow this step will result in an insufficient sample size of embryos for the EDC exposure experiment. It is also important to make sure that the hypochlorite solution is made fresh to avoid obtaining an unsynchronized population that will interfere with the ability to perform BPA exposure during early embryogenesis. Washing pellets with M9 (after hypochlorite exposure) is also a very important step. If not done properly, the high concentration of bleach might result in dead embryos. Additionally, embryos should be transferred to seeded NGM plates after 4 h. If left in the BPA solution for an extended period of time, the embryos are likely to develop into the long-lived dauer larvae. This will significantly interfere with both fecundity assay and behavioral assays. If any of these steps is missed, it is advisable to start the procedure from the very beginning. If for any reason, the NGM plate with worms is contaminated or does not have enough food, it will add stress on the worms thereby skewing the data collected. Such plates should be discarded and not be used in the experiment.
Previously, a remarkable study successfully used the C. elegans model to demonstrate that chromosome abnormalities caused by BPA result due to the breakdown of DNA repair machinery in the germline14. However, it is noteworthy that the above study design had used continuous BPA exposure of C. elegans to high doses of BPA throughout its lifespan. Our methodology was designed to study low doses of BPA exposure during early embryogenesis, so as to assay its effects on embryonic viability and subtler long-term effects on the survivors. To our knowledge, no methodology has been developed to expose early stage embryos to very low doses of BPA limited to a specified period of time; thus, making the method presented here novel.
In addition, protocols provided here can be easily adapted to study the effects of other EDCs during the proliferation phase of embryogenesis. However, in order to study late embryonic stages, the protocol will require key modifications to the critical time window of development under focus. Furthermore, our protocol will not be suitable for experiments requiring longer times of toxicant exposure, as it opens up the possibility of the alternate development pathway resulting in the long-lived dauer arrest at the second molting cycle, which is resistant to harsh conditions20. Further modifications and refinements will be needed for longer exposure times through supplement with a food source that does not have potential to metabolize the toxicant or EDC e.g. autoclaved E. coli. In conclusion, our methodology can be utilized to assess the effects of various endocrine disrupting chemicals on the fecundity and on neuronal function in the invertebrate model system C. elegans.

Exposure to environmental toxicants, particularly compounds those that tend to interfere with development, has been under careful scientific scrutiny in recent years. More than a thousand chemicals in everyday use are classified as endocrine disrupting compounds (EDC)1. Periods of rapid growth and development, which include embryonic, infant and childhood stages, have been noted to be particularly vulnerable to the deleterious effects to even low-dose EDC. Their effect have been shown to cause reproductive and neurodevelopmental disorders2. Per the US Environmental Protection Agency and US National Toxicology Program panel guidelines, a low dose may be defined as any dose below the level of one that has been reported to cause an observable biological change or damage3. Besides low-dose effects of individual EDCs, mixtures of various EDCs found at low concentrations in the environment have the potential to cause substantive cumulative effects4.
Bisphenol A (BPA or 4,4&#39;-(propane-2,2-diyl)) is a polymerizing agent found in commonly used items such as water bottles, store receipts, dental sealants, and the linings of beverage and food cans5. Due to its structural similarity to 17-&#946; Estradiol (E2) and its affinity to the ER&#945; and ER&#946; estrogen receptors, BPA has been classified as an endocrine disrupting chemical (EDC)5,6.Although weaker, BPA&#39;s affinity to estrogen receptors has been shown to affect the reproduction system of both sexes and disrupt neural functions at doses that are considered safe7,8. Changes in DNA methylation via epigenetically regulated mechanisms have been observed to cause long-term neuronal defects in mice exposed to BPA9. Specifically, BPA has also been implicated as a possible culprit for increased rates of hyperactivity, attention deficiency and increased sensitivity to drugs due to an increase seen in D1 dopamine receptors in the mouse limbic forebrain after chronic exposure9,10. Significant evidence on the deleterious effects of EDCs on human health is based on correlation studies focusing mainly on chronic exposure of populations to environmental toxicants even at low doses5; however, limitations in inferences from human studies and in manipulating experimental controls have been accepted while addressing criticisms of unsubstantiated hype11,12.
Due to the conservation of Caenorhabditis elegans&#39; genes with respect to mammals&#39;, including its steroid hormone-receptor genes, researchers have utilized this genetically tractable lab model to unravel the functional and mechanistic effects of EDCs13. Experiments with C. elegans have shown that these compounds such as BPA and BPS can cause apoptosis, embryonic lethality, disruption in the double-stranded DNA break repair mechanisms and neural function14,15,16.
Our lab has previously shown that even low-dose exposure limited to early embryogenesis leads to lowered fecundity with behavioral deficits in the surviving adults15. Habituation to a repeated stimulus is a form of non-associative learning in model systems, including C. elegans, and our methodology uses this form of non-associative learning as a behavioral output to assay the long-term effects of embryonic exposure to the toxicants17 Specifically, we provide detailed protocols for studying BPA exposure on C. elegans, including its immediate effects on embryonic lethality and long-term effects on adult behavior, with an overall schematic depicted in Figure 1. Representative results from fecundity and non-associative learning assays are provided to highlight the effectiveness of our methodology.

<table>
	<tbody>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Fisher</td>
			<td>S25761B</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Fisher</td>
			<td>BP1423-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Alfa Aesar</td>
			<td>A11470</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Fisher</td>
			<td>7487-88-9</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Fisher</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Fisher</td>
			<td>P286-1</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Fisher</td>
			<td>7758-11--4</td>
			<td></td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Fisher</td>
			<td>1310-58-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>Clorox</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Fisher</td>
			<td>BP332-500</td>
			<td></td>
		</tr>
		<tr>
			<td>LB</td>
			<td>Fisher</td>
			<td>BP1426-500</td>
			<td></td>
		</tr>
		<tr>
			<td>BPA</td>
			<td>Sigma-Aldrich</td>
			<td>239658-250g</td>
			<td></td>
		</tr>
		<tr>
			<td>BPS</td>
			<td>Sigma-Aldrich</td>
			<td>103039-100g</td>
			<td></td>
		</tr>
		<tr>
			<td>EtOH</td>
			<td>Sigma-Aldrich</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>E.coli OP50</td>
			<td>CGC</td>
			<td></td>
			<td>Repository at U of Minnesota</td>
		</tr>
		<tr>
			<td>N2 (Wild-type C. elegans) worms</td>
			<td>CGC</td>
			<td></td>
			<td>Repository at U of Minnesota</td>
		</tr>
		<tr>
			<td>Platinum wire pick</td>
			<td>Genesee Scientific</td>
			<td>59-AWP</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri plates</td>
			<td>Fisher</td>
			<td>07-202-011</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Microscope</td>
			<td>AmScope</td>
			<td>SM-2TYY</td>
			<td></td>
		</tr>
	</tbody>
</table>

long-term behavioral and reproductive consequences of embryonic exposure to low-dose toxicants

Bisphenols, such as bisphenol A (BPA) and bisphenol S (BPS) are polymerizing agents widely used in the production of plastics and numerous everyday-use products. Based on their chemical structure and estradiol-like biological properties, they have been classified as endocrine disrupting compounds (EDC). Long-term exposure to EDCs, even at low doses, has been linked to various health defects including cancer, behavioral disorders and infertility, with greater vulnerability indicated during early developmental periods. Cellular and molecular studies with the genetically tractable nematode model Caenorhabditis elegans have demonstrated that exposure to BPA causes apoptosis, embryonic lethality and disruption in the DNA repair mechanisms. We have previously reported that exposure of C. elegans embryos to low doses of different bisphenols decreases fecundity. In addition, we have shown that the effects of exposure during the very early stages of development persist into adulthood as assayed by quantifying habituation behavior, a form of non-associative learning. Here, we provide detailed protocols for embryonic exposure to low-dose EDCs as well as the associated fecundity and anterior touch habituation assays, along with representative results.

Earlier studies with C. elegans have reported that continual exposure to high BPA concentrations (&#8805;1 mM) throughout embryo development and adulthood decreases fecundity14. Subsequent studies reported from our lab have shown that embryos that have been exposed to BPA for a limited period of 4 h in the beginning stages of their development showed a decrease in the number of viable eggs laid as adults15 (Figure 2). Two key features of the methodology were that (i) the BPA concentrations used were significantly lower (0.1 &#181;M to 10 &#181;M range) and (ii) the exposure was limited to the early embryonic period. Besides decreased fecundity even at lower doses, our habituation assays revealed that worms exposed to BPA as embryos required more stimuli to become habituated when compared to worms exposed to vehicle alone15 (Figure 3). These effects are noteworthy in that they are based on low-dose BPA exposure, following the rationale that subtler effects on neuronal function that may not be morphologically apparent are likely to be discernible through behavioral changes. In short, the representative results presented here underscore the fact that the detrimental effects of BPA exposure to an embryo impacts its development, including neuronal function that can be assessed through adult behavioral assays in C. elegans. Protocols provided here can be used for testing low-dose as well as long-term effects of other potential toxicants including endocrine disruptive compounds.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56771/56771fig1.jpg" /> 
Figure 1: Schematic representation of the experimental design. Mature worms were collected and exposed to hypochlorite solution in order to collect synchronized embryos. The embryos were then exposed to different concentrations of BPA for 4 h. The exposed embryos were transferred onto seeded NGM plates, where they were allowed to grow for 60 h at 20 &#176;C. In order to test the effectiveness of exposure during the early stages of development, the L4 worms were transferred to individual plates and tested for fecundity, and young adults were tested with anterior touch habituation. <a href="https://cloudflare.jove.com/files/ftp_upload/56771/56771fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56771/56771fig2.jpg" /> 
Figure 2: Fecundity is lowered by BPA. Exposure to 1 &#181;M and higher concentrations of BPA significantly decreased the number of eggs laid compared to the control (represented by the horizontal line; n = 10, *p &#60;0.05). Error bars denote SEM. Analysis done using ANOVA, Tukey&#39;s post hoc test. This figure has been modified from Mersha et al., 201515. <a href="https://cloudflare.jove.com/files/ftp_upload/56771/56771fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56771/56771fig3.jpg" /> 
Figure 3: Long-term effects of embryonic BPA exposure are reflected in adult behavior. Embryos exposed to BPA concentrations as low as 0.1 &#181;M affects non-associative anterior touch habituation in adults (n = 60, *p &#60;0.05). Error bars denote SEM; ANOVA, Tukey&#39;s post hoc test. This figure has been modified from Mersha et al., 201515. <a href="https://cloudflare.jove.com/files/ftp_upload/56771/56771fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Long-term Behavioral and Reproductive Consequences of Embryonic Exposure to Low-dose Toxicants at JoVE.com

Long-term Behavioral and Reproductive Consequences of Embryonic Exposure to Low-dose Toxicants

Exposure to environmental toxicants can acutely impact development with long-term effects. Detailed protocols are provided to illustrate a strategy using an effective lab model to study the effect of early embryonic exposure to bisphenol A. We provide fecundity and behavioral assays to monitor the effectiveness of our toxicant exposure bio-assays.

Bisphenols, such as bisphenol A (BPA) and bisphenol S (BPS) are polymerizing agents widely used in the production of plastics and numerous everyday-use ...

long-term-behavioral-reproductive-consequences-embryonic-exposure-to

Research

JoVE Journal

Developmental Biology

6.0K Views.  Johns Hopkins School of Medicine. Exposure to environmental toxicants can acutely impact development with long-term effects. Detailed protocols are provided to illustrate a strategy using an effective lab model to study the effect of early embryonic exposure to bisphenol A. We provide fecundity and behavioral assays to monitor the effectiveness of our toxicant exposure bio-assays.

Video: Long-term Behavioral and Reproductive Consequences of Embryonic Exposure to Low-dose Toxicants

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms including wound healing disorder. The physiological process of wound healing is highly complex. The formation of granulation tissue is a key step in this process resulting in a preliminary wound closure and providing a network of new capillary blood vessels &#8211; either through vasculogenesis (novel formation) or angiogenesis (sprouting of existing vessels). Both vasculo- and angiogenesis require functional, directed migration of endothelial cells. Thus, investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of chemical induced wound healing disorders and to potentially identify novel strategies for therapeutic intervention.
We assessed impaired wound healing after alkylating agent exposure and tested potential candidate compounds for treatment. We used a set of techniques outlined in this protocol. A modified Boyden chamber to quantitatively investigate chemokinesis of EEC is described. Moreover, the use of the wound healing assay in combination with track analysis to qualitatively assess migration is illustrated. Finally, we demonstrate the use of the fluorescent dye TMRM for the investigation of mitochondrial membrane potential to identify underlying mechanisms of disturbed cell migration. The following protocol describes basic techniques that have been adapted for the investigation of EEC.

Investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of certain illnesses and to potentially identify novel strategies for therapeutic intervention. The following protocol describes techniques to assess cell migration that have been adapted for the investigation of EEC.

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms ...

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

A Versatile Mounting Method for Long Term Imaging of Zebrafish Development

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. In particular, posterior body elongation is an essential process in embryonic development by which multiple tissue deformations act together to direct the formation of a large part of the body axis. In order to observe this process by long-term time-lapse imaging it is necessary to utilize a mounting technique that allows sufficient support to maintain samples in the correct orientation during transfer to the microscope and acquisition. In addition, the mounting must also provide sufficient freedom of movement for the outgrowth of the posterior body region without affecting its normal development. Finally, there must be a certain degree in versatility of the mounting method to allow imaging on diverse imaging set-ups. Here, we present a mounting technique for imaging the development of posterior body elongation in the zebrafish D. rerio. This technique involves mounting embryos such that the head and yolk sac regions are almost entirely included in agarose, while leaving out the posterior body region to elongate and develop normally. We will show how this can be adapted for upright, inverted and vertical light-sheet microscopy set-ups. While this protocol focuses on mounting embryos for imaging for the posterior body, it could easily be adapted for the live imaging of multiple aspects of zebrafish development.

Here, we present a versatile mounting method that allows for the long-term time-lapse imaging of the posterior body development of live zebrafish embryos without perturbing normal development.

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. ...

A Simple Chamber for Long-term Confocal Imaging of Root and Hypocotyl Development

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and subcellular processes, high resolution time-lapse microscopy strategies that preserve physiological conditions are required. Plant tissues must have adequate nutrient and water supply with sustained gaseous exchange but, when submerged and immobilized under a coverslip, they are particularly susceptible to anoxia. One strategy that has been successfully employed is the use of a perfusion system to maintain a constant supply of oxygen and nutrients. However, such arrangements can be complicated, cumbersome, and require specialized equipment. Presented here is an alternative strategy for a simple imaging system using perfluorodecalin as an immersion medium. This system is easy to set up, requires minimal equipment, and is easily mounted on a microscope stage, allowing several imaging chambers to be set up and imaged in parallel. In this system, lateral root growth rates are indistinguishable from growth rates under standard conditions on agar plates for the first two days, and lateral root growth continues at reduced rates for at least another day. Plant tissues are supplied with nutrients via an agar slab that can be used also to administer a range of pharmacological compounds. The system was established to monitor lateral root development but is readily adaptable to image other plant organs such as hypocotyls and primary roots.

Presented here is a simple technique for high-resolution confocal time-lapse imaging of root and hypocotyl development for up to 3 days using high numerical-aperture objectives and perfluorodecalin as an immersion medium.

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and ...

Long-term Live Imaging of Drosophila Eye Disc

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have been used to study many biological processes, such as cell proliferation, differentiation, growth, migration, apoptosis, competition, cell-cell signaling, and compartmental boundary formation. However, methods for the long-term ex vivo culture and live imaging of the imaginal discs have not been satisfactory, despite many efforts. Recently, we developed a method for the long-term ex vivo culture and live imaging of imaginal discs for up to 18 h. In addition to using a high insulin concentration in the culture medium, a low-melting agarose was also used to embed the disc to prevent it from drifting during the imaging period. This report uses the eye-antennal discs as an example. Photoreceptor R3/4-specific m&#948;0.5-Ga4 expression was followed to demonstrate that photoreceptor differentiation and ommatidial rotation can be observed during a 10 h live imaging period. This is a detailed protocol describing this simple method.

Long-term Live Imaging of Drosophila Eye Disc

This protocol describes a detailed method for the long-term ex vivo culture and live imaging of a Drosophila imaginal disc. It demonstrates photoreceptor differentiation and ommatidial rotation within the 10 h period of live imaging of the eye disc. The protocol is simple and does not require expensive setup.

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have ...

Collection of Post-mating Semen from the Female Reproductive Tract and Measurement of Semen Liquefaction in Mice

In mice, ejaculated semen is deposited in the uterus. After ejaculation, the semen changes consistency from gel-like to watery, a process called liquefaction. In this study, we show how to collect the post-ejaculated semen from the female reproductive tract in a mouse model. First, adult female mice in the estrus stage were housed in a male&#39;s cage overnight. The next morning, copulation was confirmed by the presence of copulatory plug at the vaginal opening. Female mice with copulatory plugs were euthanized, and each reproductive tract was collected as a whole (vagina, uterus, oviducts, ovaries), ensuring a closed system to contain the semen. The reproductive tract was placed in a 1.5 mL microcentrifuge tube, and the vagina was cut off to release the semen into the tube. To ensure maximum semen volume for analysis, toothless forceps were used to squeeze the uterine horns from ovarian end to vaginal end expelling remaining semen. The whole reproductive tract was then discarded. The semen-containing tube was briefly spun down. A 25 &#956;L capillary pipette was placed into the tube at a 180&#176; angle (parallel to the tube wall). The amount of time used to fill the capillary tube to the 25 &#956;L line was recorded. Semen from a proven male breeder usually takes approximately 60-180 s to fill a 25 &#956;L capillary tube. This semen collection technique can also be used in other downstream applications such as sperm imaging and motility analysis.

Semen liquefaction is required for sperm to be liberated from the seminal gel. This study provides the procedures for collecting semen from the female reproductive tract post-coitus, and measuring semen liquefaction time.

In mice, ejaculated semen is deposited in the uterus. After ejaculation, the semen changes consistency from gel-like to watery, a process called ...

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...