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. 2022 Mar 20;12(6):e4351.
doi: 10.21769/BioProtoc.4351.

TUNEL Labeling to Detect Double-stranded DNA Breaks in Caenorhabditis elegans Gonads

Peter A Kropp  1 , Kyle Rhodehouse  1 , Andy Golden  1
Affiliations

TUNEL Labeling to Detect Double-stranded DNA Breaks in Caenorhabditis elegans Gonads

Peter A Kropp et al. Bio Protoc. .

Abstract

Analysis of DNA double strand breaks (DSBs) is important for understanding dyshomeostasis within the nucleus, impaired DNA repair mechanisms, and cell death. In the C. elegans germline, DSBs are important indicators of all three above-mentioned conditions. Although multiple methods exist to assess apoptosis in the germline of C. elegans, direct assessment of DSBs without the need for a reporter allele or protein-specific antibody is useful. As such, unbiased immunofluorescent approaches can be favorable. This protocol details a method for using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) to assess DNA DSBs in dissected C. elegans germlines. Germlines are co-labeled with DAPI to allow for easy assessment of DNA DSBs. This approach allows for qualitative or quantitative measures of DNA DSBs. Graphic abstract: Schematic for TUNEL labeling of C. elegans germlines.

Keywords: C. elegans; Double strand breaks; Germline; TUNEL.

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Conflict of interest statement

Competing interestsThe authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Picking animals from plate to slide.
(A) Using a platinum wire pick, pick the animal(s) from the culture plate to a watch glass containing M9 buffer. (B) Using a sweeping motion, gently remove the animal(s) from the pick into the M9 buffer in the watch glass. (C) Using a sweeping motion, gently lift individual animals from the watch glass and transfer them to a slide containing a drop of M9 buffer. (D) Representative schematic of a slide with an individual animal contained within a droplet of M9 buffer on a dried bed of poly-L-lysine. Representations are not to scale.
Figure 2.
Figure 2.. Gonad dissection from adult C. elegans.
(A) Representative image through a dissecting microscope of a day-one adult C. elegans with a 23 G 1¼ needle for scale. The tip of the needle is adjacent to the junction of the pharynx and intestine (also indicated by change from light to dark color). This is where the incision should be made. (B) Representative image of an extruded gonad as viewed through a dissecting microscope. The intestine will also extrude. Note that the head was not completely removed in this instance. (C) Enhanced image (from B) of extruded day-one adult gonad with regions labeled. MZ: Mitotic zone; TZ: Transition zone. Scale bars (A and B): 500 µm; (C) 100 µm.
Figure 3.
Figure 3.. Schematic for fixing, washing, and staining extruded gonads.
The animals will be contained within a droplet of buffer. Pipet or wick away (with a KimWipe) buffers at the end of each step. Add buffers with a pipet at the beginning of each step. To avoid disrupting the samples, pipet or wick from the edge of the buffer droplet as indicated by the area in the red oval. If necessary, tilt the slide to make wicking easier. Representations not to scale.
Figure 4.
Figure 4.. Representative maximum intensity projection of a wild-type L4 C. elegans gonad co-labeled with DAPI and TUNEL.
DAPI-stained nuclei are labeled in blue and TUNEL-positive nuclei are labeled in green. As this is an L4 gonad, no oocytes are present. The different regions of the gonad are discernable based on nuclear (DAPI) structure. Due to crossover formation during meiosis (diplotene), nuclei will label with TUNEL. Artifactual labeling of mitotic nuclei with TUNEL has been reported in other systems, including human tissue sections (Labat-Moleur et al., 1997) and Drosophila ovaries (Qi and Calvi, 2016). DNA nicks during replication are likely the cause (Mets and Meyer, 2009). Apparent pale labeling in the middle pachytene region is an artifact from stitching two z-stacks into a single image. Scale bar: 100 μm.
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