Evidence that single-stranded DNA breaks are a normal feature of koala sperm chromatin, while double-stranded DNA breaks are indicative of DNA damage

Abstract

In this study, we have used single and double comet assays to differentiate between single-and double-stranded DNA damage in an effort to refine the interpretation of DNA damage in mature koala spermatozoa. We have also investigated the likelihood that single-stranded DNA breakage is part of the natural spermiogenic process in koalas, where its function would be the generation of structural bends in the DNA molecule so that appropriate packaging and compaction can occur. Koala spermatozoa were examined using the sperm chromatin dispersion test (SCDt) and comet assays to investigate non-orthodox double-stranded DNA. Comet assays were conducted under 1) neutral conditions; and 2) neutral followed by alkaline conditions (double comet assay); the latter technique enabled simultaneous visualisation of both single-stranded and double-stranded DNA breaks. Following the SCDt, there was a continuum of nuclear morphotypes, ranging from no apparent DNA fragmentation to those with highly dispersed and degraded chromatin. Dispersion morphotypes were mirrored by a similar diversity of comet morphologies that could be further differentiated using the double comet assay. The majority of koala spermatozoa had nuclei with DNA abasic-like residues that produced single-tailed comets following the double comet assay. The ubiquity of these residues suggests that constitutive alkali-labile sites are part of the structural configuration of the koala sperm nucleus. Spermatozoa with 'true' DNA fragmentation exhibited a continuum of comet morphologies, ranging from a more severe form of alkaline-susceptible DNA with a diffuse single tail to nuclei that exhibited both single-and double-stranded breaks with two comet tails.

Osmotic stress and cryoinjury of koala sperm: an integrative study of the plasma membrane, chromatin stability and mitochondrial function

“…Similarly, Breed et al (2001) have also noted that the koala sperm nucleus is more prone to dispersion with Triton X-100 detergent than wombat spermatozoa, and this may be related to the presence of a large nuclear vacuole in the base of the koala sperm nucleus, which is not apparent in wombat. With respect to the second possibility, we have previously shown and proposed the notion that alkali-labile sites (ALS) present on the DNA (Zee et al 2009b) facilitate the DNA bending necessary to configure the tertiary and ultimate structure of the DNA-protamine complex . These ALS could be present in the mature spermatozoa in the form of unpaired single-stranded DNA stretches or simply as apurinic or apyrimidinic sites.…”

“…These ALS could be present in the mature spermatozoa in the form of unpaired single-stranded DNA stretches or simply as apurinic or apyrimidinic sites. This suggestion was made on the basis of the prominent presence of alkaline comets in normal spermatozoa (Zee et al 2009b). It is possible that further structural damage and/ or oxidative attack to these potentially weakened areas may lead to chromatin relaxation.…”

“…For example, koala spermatozoa have been shown to have a post-thaw viability of up to 60-70% if frozen in high levels of glycerol (14% v/v) combined with a relatively slow freezing rate of 6 8C/min. However, extended incubation of thawed koala spermatozoa for a further 2 h rapidly reduces the percentage of live spermatozoa and results in a corresponding increase in the number of cells with swollen or relaxed chromatin (Zee et al 2008(Zee et al , 2009b. It is uncertain whether this modified form of chromatin is either a consequence of physical swelling or distortion of the DNA/protamine complex in response to osmotically induced injury or is associated with an increase in the fragmentation of the DNA molecule , Zee et al 2009b.…”

“…Mitochondrial membrane potential (MMP) has been shown to be a sensitive indicator of the bioenergetic functions within the mitochondrion (Duchen 2004), and while Zee et al (2007Zee et al ( , 2009aZee et al ( , 2009b have previously used the JC1 probe (5,5 0 ,6,6 0 -tetrachloro-1,1 0 ,3,3 0 -tetraethylbenzimidazolyl carbocyanine iodide) to investigate the effect of cryoinjury on the koala sperm midpiece, the relative detrimental effects of osmotic stress vs structural damage associated with intracellular ice crystal damage remain unresolved. While the response of spermatozoa to the damaging effects of osmotic stress during cryopreservation can be demonstrated artificially by exposure of spermatozoa to a range of osmotically defined media (Curry & Watson 1994, Meyers 2005, Willoughby et al 1996, to our knowledge, this experimental model has never been used before to examine the effect of osmosis on sperm DNA, and certainly not for marsupial sperm chromatin.…”

Cytometric solutions in veterinary andrology: Developments, advantages, and limitations

“…Moreover, dependent on species, fragmented DNA as such does not necessarily indicate sperm damage or dysfunction. A recent study (73) demonstrated that the koala sperm head contains a massive amount of single strand DNA breaks, but they appear to be a normal feature for the species.…”