A C. elegans screening platform for the rapid assessment of chemical disruption of germline function

Abstract

Background: Despite the developmental impact of chromosome segregation errors, we lack the tools to assess environmental effects on the integrity of the germline in animals.

Objectives: We developed an assay in Caenorhabditis elegans that fluorescently marks aneuploid embryos after chemical exposure.

Methods: We qualified the predictive value of the assay against chemotherapeutic agents as well as environmental compounds from the ToxCast Phase I library by comparing results from the C. elegans assay with the comprehensive mammalian in vivo end point data from the ToxRef database.

Results: The assay was highly predictive of mammalian reproductive toxicities, with a 69% maximum balanced accuracy. We confirmed the effect of select compounds on germline integrity by monitoring germline apoptosis and meiotic progression.

Conclusions: This C. elegans assay provides a comprehensive strategy for assessing environmental effects on germline function.

Figure 1

Design of the screening platform. (A) Worms from the aneuploidy-reporting Pxol-1::GFP strain are exposed to libraries of environmental compounds for either 24 hr or 65 hr. After exposure, the induction of aneuploidy can be visualized and quantified by fluorescence microscopy (B; bar = 100 µm) or automated detection and sorting of the worms (C). In B, several embryos expressing GFP (GFP+) can clearly be visualized. C shows automated reading of the embryos. A population of GFP+ embryos can be detected as distinct from GFP– embryos and debris, which appear below the black bar.

Figure 2

Chemical induction of aneuploidy in C. elegans. (A) Pxol-1::GFP worms were exposed to 100 µm nocodazole or 0.1% DMSO (control) for 24 hr. Two GFP+ embryos are visible within the nocodazole-treated worm’s uterus (arrows) adjacent to the autofluorescence emanating from the gut; bar = 50 µm. (B) Embryonic viability (mean percent ± SE) after either DMSO or nocodazole exposure. (C) Chemotherapeutic screen; the worms were exposed for 24 hr or 65 hr to 100 µM of each compound. The number of GFP+ embryos per worm was recorded, corrected for the average number of embryos found in each worm, and expressed as the log fold ratio over DMSO (mean percent ± SE; two-tailed Mann–Whitney U test, 95% CI, chosen over ANOVA with post-test correction to test for significant differences for each compound over DMSO because of high differences in sample variance). Each chemical exposure was performed six times. ^aLethal at 65 hr. *p ≤ 0.05, and **p ≤ 0.01, by two-tailed Mann–Whitney U test, 95% CI.

Figure 3

Screening of environmental chemicals. Worms were exposed for either 24 hr (A) or 65 hr (B) to each compound, at a concentration of 100 µM [except for mancozeb, dicofol, 2-(thiocyanomethylthio) benzothiazole (TCMTB), phosalone, chlorophene, endosulfan, parathion-methyl, which were further diluted 10-fold, and chlorpyrifos-methyl, which was used at 1 µM, to circumvent lethality]. The number of green embryos per worm was recorded and corrected for the average number of embryos found in each worm. The number was then expressed as the log fold ratio over DMSO. Chemicals are listed in order in Supplemental Material, Tables S2 and S3 (http://dx.doi.org/10.1289/ehp.1206301). The compounds were categorized according to their assessed mammalian reproductive toxicity [i.e., the number of mammalian end points for which they were positive: high reproductive toxicity (> 2 end points), intermediate reproductive toxicity (1 end point), and no reproductive toxicity (0 end points)]. At 65 hr, the mean value of fold-induction for the high and intermediate reproductive toxicity groups was significantly higher than the no–reproductive toxicity group (p = 0.008). Each chemical was tested three times.

Figure 4

Functional validation of selected compounds. (A) Germline apoptosis assay quantification. After exposure to each of the 10 least aneugenic compounds in the C. elegans assay or to each of the 10 most aneugenic compounds, we quantified apoptotic levels through the use of the Plim-7ced-1::gfp reporter DMSO was the negative control, and the DNA damaging agent camptothecin the positive control. The lower and upper edges of the box plot represent the first and third quartiles, respectively, with the median represented as a line within the box; the whiskers extend to ± 1.5 times the interquartile range. (B) DAPI staining of germ­line nuclei revealed profound germ­line defects after exposure to Maneb and TCMTB (bars = 50 µm). Assembled germ­lines show areas of reduced nuclear density (asterisks), intermixed meiotic stages (red arrowheads), and unequally spaced diakinetic nuclei (white arrows). Insets show high magnification examples of late diakinetic nuclei (bars = 4 µm); note that nuclei with chromosomes in a pachytene stage-like organization are present intermixed with diakinetic nuclei in late prophase after maneb exposure. *p < 0.001 compared with DMSO by ANOVA followed by Dunn’s comparison. The numbers of apoptotic nuclei in the 10 least and most aneugenic compounds were highly significantly different from each other (p < 0.0001; two-tailed Mann–Whitney U test, 95% CI)

Figure 5

High throughput detection of genetic and chemical induction of aneuploidy. (A) Overlay of bright field and fluorescence images of a Pxol-1::GFP, him-8 worm with two embryos; the GFP⁺ embryo is clearly distinguishable from the GFP^– embryo next to it (bar = 100 µm). (B) Automated detection of GFP⁺ embryos using the COPAS BIOSORT; the black line represents the GFP⁺ threshold as determined by Pxol-1::GFP without him-8 (WT count). The GFP⁺ population of embryos is more abundant in nocodazole-treated worms compared with DMSO-treated worms in both the Pxol-1::GFP and the Pxol-1::GFP; col-121 (sensitized) backgrounds.