Interpreting sperm DNA damage in a diverse range of mammalian sperm by means of the two-tailed comet assay

Abstract

Key ConceptsThe two-dimensional Two-Tailed Comet assay (TT-comet) protocol is a valuable technique to differentiate between single-stranded (SSBs) and double-stranded DNA breaks (DSBs) on the same sperm cell.Protein lysis inherent with the TT-comet protocol accounts for differences in sperm protamine composition at a species-specific level to produce reliable visualization of sperm DNA damage.Alkaline treatment may break the sugar-phosphate backbone in abasic sites or at sites with deoxyribose damage, transforming these lesions into DNA breaks that are also converted into ssDNA. These lesions are known as Alkali Labile Sites "ALSs."DBD-FISH permits the in situ visualization of DNA breaks, abasic sites or alkaline-sensitive DNA regions.The alkaline comet single assay reveals that all mammalian species display constitutive ALS related with the requirement of the sperm to undergo transient changes in DNA structure linked with chromatin packing.Sperm DNA damage is associated with fertilization failure, impaired pre-and post- embryo implantation and poor pregnancy outcome.The TT is a valuable tool for identifying SSBs or DSBs in sperm cells with DNA fragmentation and can be therefore used for the purposes of fertility assessment. Sperm DNA damage is associated with fertilization failure, impaired pre-and post- embryo implantation and poor pregnancy outcome. A series of methodologies to assess DNA damage in spermatozoa have been developed but most are unable to differentiate between single-stranded DNA breaks (SSBs) and double-stranded DNA breaks (DSBs) on the same sperm cell. The two-dimensional Two-Tailed Comet assay (TT-comet) protocol highlighted in this review overcomes this limitation and emphasizes the importance in accounting for the difference in sperm protamine composition at a species-specific level for the appropriate preparation of the assay. The TT-comet is a modification of the original comet assay that uses a two dimensional electrophoresis to allow for the simultaneous evaluation of DSBs and SSBs in mammalian spermatozoa. Here we have compiled a retrospective overview of how the TT-comet assay has been used to investigate the structure and function of sperm DNA across a diverse range of mammalian species (eutheria, metatheria, and prototheria). When conducted as part of the TT-comet assay, we illustrate (a) how the alkaline comet single assay has been used to help understand the constitutive and transient changes in DNA structure associated with chromatin packing, (b) the capacity of the TT-comet to differentiate between the presence of SSBs and DSBs (c) and the possible implications of SSBs or DSBs for the assessment of infertility.

Keywords: Sperm DNA damage; eutheria; fertility; male factor; mammalian reproduction; metatheria; prototheria.

Figure 1

Single neutral and alkaline comets. (A) Neutral comet from rhinoceros (Rhinoceros sondaicus) sperm showing a tail of mobilized DNA fragments (right direction of the image) as a consequence of double strand DNA breaks (DSBs) at the origin of the sperm head. Sperm without comet tail (labeled as normal for this condition) do not contain detectable levels of DSBs, but display a small halo of compact chromatin. (B) Alkaline comet of rhinoceros sperm (pink comet) showing a tail of mobilized DNA fragments which are interpreted as single strand DNA stretches derived from short DSBs susceptible to be denatured, single breaks (SSBs) and alkali labile associated with structural comets (pseudo-colored green). Magnified regions (box) within the neutral (C) and an alkaline comet tail (D) are provided to visualize the difference in the chromatin structure along with filtered images to enhance differences in chromatin texture (E—neutral and F—alkaline).

Figure 2

Structural alkaline comets in different mammalian sperm (A–J) eutherian species, (K,L) metatherian species and (M) a prototherian species.

Figure 3

Visualization of the alkali labile sites (ALS) using DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) as a structural feature of the spermatozoa and peripheral blood leukocytes in different mammal species. High alkali denaturation conditions (DBD-FISH High) show most of the ALS present al the spermatozoa. Mild alkaline conditions (DBD-FISH Low) revealed the localization of regions most sensitive to alkaline denaturation.

Figure 4

(A) Stallion structural comets showing differences in the density of DNA but similar tail length; (B) Density profile of horse comets showing differences in fluorescence intensity—different colored circles correspond to the colored graphic profiles of individual spermatozoa; (C) Structural (black dot) and damaged (red dot) koala spermatozoa showing differences in the amount of DNA and DNA migration distance; (D) Density profile of koala comets showing differences in fluorescence intensity and length of comet tail.

Figure 5

Original (A) and digitally enhanced image (B) to show whole DNA fragment distribution DNA map following a two tailed comet assay in a human sperm cell. The first neutral electrophoresis results in a horizontal migration along the X axis of DNA fragments formed as a consequence of DSBs. After turning the microgel 90°, the second electrophoresis results in the migration of DNA along the vertical Y axis and is conducted under alkaline conditions; the alkaline comet assay reveals both structural Alkali Labile Sites (ALSs) and “true” SSBs that have elongated comet tails. The green fluorescence is associated with proteinaceous remnants of the sperm head and flagellum.

Figure 6

Pseudo-colored images of sperm DNA following a TT comet assay and illustrating different comet morphology associated with different types of DNA damage. (A) Type 1 (yellow)—structural comet; Type 2 (pink)—SSBs plus ALSs; Type 3 (blue) DDBs, SSBs plus ALS within the same TT comet and (B) Type 4 (gray)—DSBs and structural comet.

Figure 7

TT comet produced after controlled double strand DNA cleavage using the restriction enzyme Alu-I (AG_CT). Original image (A), and panel electronically enhanced (B). R: different regions identified at the TT-comet. See text for detailed explanation.