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

Allard P, Kleinstreuer NC, Knudsen TB, Colaiácovo MP - Environ. Health Perspect. (2013)

Bottom Line: Despite the developmental impact of chromosome segregation errors, we lack the tools to assess environmental effects on the integrity of the germline in animals.We confirmed the effect of select compounds on germline integrity by monitoring germline apoptosis and meiotic progression.This C. elegans assay provides a comprehensive strategy for assessing environmental effects on germline function.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115 , USA.

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.

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Related in: MedlinePlus

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.
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f1: 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.

Mentions: We developed a chemical strategy using the Pxol-1::gfp strain (Figure 1). Specifically, liquid cultures of the strain are exposed to chemicals of interest at 100 µM, a concentration commonly used in chemical screens in C. elegans (Boyd et al. 2010a, 2010b). The worm germline consists of nuclei simultaneously moving from the distal to the proximal end of the gonad and progressing through the meiotic stages in a synchronous manner. This establishes a spatial and temporal gradient of meiotic progression in C. elegans, with a well-characterized timing of events (Jaramillo-Lambert et al. 2007; Pazdernik and Schedl 2013). Consequently, we exposed the worms for durations of 24 hr and 65 hr in order to capture the effects of exposure at distinct stages of germline progression. Aneuploidies generated after a 24-hr test interval arise from the impairment of late meiotic (late pachytene and onward) and early embryonic processes, whereas the 65-hr interval captures aneuploidies originating from the disruption of any mitotic and meiotic events in the germline in addition to early embryonic stages. After exposure, the worms were readily observed under a fluorescence microscope. The number of GFP+ embryos were counted and normalized to the total number of embryos present to correct for decreased embryo production. We also established the automated detection and sorting of the GFP+ worms by using the COPAS BIOSORT (Union Biometrica) for sorting of viable worms and embryos. The use of a flow cytometry sorting system allowed us to scale up the numbers of chemicals being tested and the speed of screening, thus enabling high throughput capability (see below).


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

Allard P, Kleinstreuer NC, Knudsen TB, Colaiácovo MP - Environ. Health Perspect. (2013)

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.
© Copyright Policy - public-domain
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672921&req=5

f1: 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.
Mentions: We developed a chemical strategy using the Pxol-1::gfp strain (Figure 1). Specifically, liquid cultures of the strain are exposed to chemicals of interest at 100 µM, a concentration commonly used in chemical screens in C. elegans (Boyd et al. 2010a, 2010b). The worm germline consists of nuclei simultaneously moving from the distal to the proximal end of the gonad and progressing through the meiotic stages in a synchronous manner. This establishes a spatial and temporal gradient of meiotic progression in C. elegans, with a well-characterized timing of events (Jaramillo-Lambert et al. 2007; Pazdernik and Schedl 2013). Consequently, we exposed the worms for durations of 24 hr and 65 hr in order to capture the effects of exposure at distinct stages of germline progression. Aneuploidies generated after a 24-hr test interval arise from the impairment of late meiotic (late pachytene and onward) and early embryonic processes, whereas the 65-hr interval captures aneuploidies originating from the disruption of any mitotic and meiotic events in the germline in addition to early embryonic stages. After exposure, the worms were readily observed under a fluorescence microscope. The number of GFP+ embryos were counted and normalized to the total number of embryos present to correct for decreased embryo production. We also established the automated detection and sorting of the GFP+ worms by using the COPAS BIOSORT (Union Biometrica) for sorting of viable worms and embryos. The use of a flow cytometry sorting system allowed us to scale up the numbers of chemicals being tested and the speed of screening, thus enabling high throughput capability (see below).

Bottom Line: Despite the developmental impact of chromosome segregation errors, we lack the tools to assess environmental effects on the integrity of the germline in animals.We confirmed the effect of select compounds on germline integrity by monitoring germline apoptosis and meiotic progression.This C. elegans assay provides a comprehensive strategy for assessing environmental effects on germline function.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115 , USA.

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.

Show MeSH
Related in: MedlinePlus