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

Abstract

This study investigated whether cryopreservation-induced injury to koala spermatozoa could be explained using an experimental model that mimics the structural and physiological effects of osmotic flux. DNA labelling after in situ nick translation of thawed cryopreserved spermatozoa revealed a positive correlation (rZ0.573; P!0.001; nZ50) between the area of relaxed chromatin in the nucleus and the degree of nucleotide labelling. While the chromatin of some spermatozoa increased more than eight times its normal size, not all sperm nuclei with relaxed chromatin showed evidence of nucleotide incorporation. Preferential staining associated with sperm DNA fragmentation (SDF) was typically located in the peri-acrosomal and peripheral regions of the sperm head and at the base of the spermatozoa where it appear to be 'hot spots' of DNA damage following cryopreservation. Results of the comparative effects of anisotonic media and cryopreservation on the integrity of koala spermatozoa revealed that injury induced by exposure to osmotic flux, essentially imitated the results found following cryopreservation. Plasma membrane integrity, chromatin relaxation and SDF appeared particularly susceptible to extreme hypotonic environments. Mitochondrial membrane potential (MMP), while susceptible to extreme hypo-and hypertonic environments, showed an ability to rebound from hypertonic stress when returned to isotonic conditions. Koala spermatozoa exposed to 64 mOsm/kg media showed an equivalent, or more severe, degree of structural and physiological injury to that of frozen-thawed spermatozoa, supporting the hypothesis that cryoinjury is principally associated with a hypo-osmotic effect. A direct comparison of SDF of thawed cryopreserved spermatozoa and those exposed to a 64 mOsm/kg excursion showed a significant correlation (rZ0.878; P!0.05; nZ5); however, no correlation was found when the percentage of sperm with relaxed chromatin was compared. While a cryo-induced osmotic injury model appears to explain post-thaw changes in koala SDF, the mechanisms resulting in relaxed chromatin require further study. A lack of correlation between the percentage of sperm with relaxed chromatin and SDF suggests that the timing of these pathologies are asynchronous. We propose an integrative model of cryo-induced osmotic injury that involves a combination of structural damage (rupture of membrane) and oxidative stress that first leads to the reduction of MMP and the relaxation of chromatin, which is then ultimately followed by an increase in DNA fragmentation.

Amphibian sperm DNA fragmentation

“…In these species it is possible that the nuclear basic proteins of the spermatozoon render the DNA more susceptible to protein depletion (Zee et al 2009). The chromatin-swelling phenomenon observed here is similar to that described in the koala (Johnston et al 2006;2012a), common wombat (Vombatus ursinus; Johnston et al 2006) and fish ). Chromatin swelling in African clawed frog spermatozoa without DNA fragmentation may be due to the extensive presence of alkali labile sites (ALS), highly sensitive DNA motifs (possible regions of specific repetitive sequences; Cortés-Gutiérrez et al…”

“…Gosálvez et al (2010), suggest that the incidence of increased single-and/or double-stranded DNA breaks following cryopreservation is not a direct consequence of the freezing process, but rather, that modifications to the chromatin-SNBP interaction as a result of freeze-thawing predisposes the sperm DNA to fragmentation that manifests, not immediately, but over time following thawing. In most mammalian species, including but not limited to the koala (Johnston et al 2012a), stallion ), ram and Asian elephant (Elephas maximus; Imrat et al 2012), the basal level of sperm DNA damage observed immediately after thawing does not differ significantly from that observed in fresh spermatozoa. However, the chromatin stability tends to deteriorate more rapidly following thawing and incubation and can substantially limit the longevity of the DNA.…”

“…Although a number of studies have demonstrated the damaging effects of aniso-osmotic media on spermatozoa in terms of plasma membrane integrity and motility (Blasse et al 2010), few have examined the effect of osmotic stress on sperm DNA. Johnston et al (2012a) recorded significant increases in the percentage of koala spermatozoa with fragmented DNA following exposure to both hypo-osmotic and hyper-osmotic media. Furthermore, the authors reported a significant correlation in DNA fragmentation between cryopreserved-thawed spermatozoa and those exposed to a hypo-osmotic media.…”

“…During cryopreservation, the formation of extracellular ice crystals forms a hypertonic environment resulting in the outflow of water across the plasma membranes of the spermatozoa and a decrease in cellular volume; the removal of cryoprotectant upon thawing restores the osmotic environment, causing a further influx of water and further damage to the sperm cell (Johnston et al 2012a). Although a number of studies have demonstrated the damaging effects of aniso-osmotic media on spermatozoa in terms of plasma membrane integrity and motility (Blasse et al 2010), few have examined the effect of osmotic stress on sperm DNA.…”

New insights into the reproductive physiology and management of the female koala (Phascolarctos cinereus): factors affecting the control of the oestrous cycle

“…Despite significant advances in koala sperm cryopreservation (Johnston et al, 2006, Zee et al, 2007, Johnston et al, 2012, Johnston et al, 2013c, the quality of cryopreserved koala semen after thawing is still inadequate for successful AI. Although this precludes genome resource banking for the moment, chilled semen storage offers significant 1-5 logistical flexibility for the application of AI in the koala, particularly as it relates to the national and global movement of genetic material.…”