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* To whom correspondence should be addressed. E-mail: s.johnston1{at}uq.edu.au.
Koala sperm nuclei show a tendency to swell following cryopreservation but it is uncertain whether this phenomenon is associated with DNA fragmentation. In this study, a modified version of the sperm chromatin dispersion test (SCDt) was validated for use with koala spermatozoa and is the first use of the test for a marsupial. Cryopreserved spermatozoa (multiple straws) from a single koala were used to explore the relationship between sperm morphology, viability, chromatin dispersion and DNA fragmentation. A SCDt prototype kit (Sperm Halomax) was specifically developed for koala spermatozoa using a lysing solution that did not contain dithiotheitol (DTT). DNA fragmentation of lysed and non-lysed spermatozoa was examined in microgel slides and validated by means of in situ nick translation (ISNT). The SCDt was then applied to the analysis of extended and frozen-thawed semen samples of three different koalas. Spermatozoa were classified into three distinct morphotypes following the SCDt; (1) KSM-1 - rod-shaped cells with no halo of DNA; (2) KSM-2 - round shaped nuclei with various degrees of halo formation about a dense chromatin core and (3) KSM-3 - rod or rounded shaped nuclei consisting of an inner chromatin core but with large disperse halos of stellar chromatin. While ISNT following the SCDt did not label the KSM-1 morphotype, both KSM-2 and KSM-3 morphotypes stained positively for DNA fragmentation. ISNT was not able to differentiate between KSM-2 and KSM-3 morphotypes. While application of the SCDt to the spermatozoa of another 3 koalas, showed that there was no difference in the percentage of the three sperm morphotypes found between extended and frozen-thawed semen, thawed spermatozoa incubated at 35oC for 2h showed an increase in the incidence of KSM-3 and a corresponding decrease in KSM-2. We propose that KSM-1 and KSM-2 morphotypes represent nuclei that show either no, or only limited, sperm DNA fragmentation respectively. It is likely that the halos formed around KSM-2 morphotypes are from DNA that is damaged as part of the normal processing of the spermatozoa and is a consequence of the lack of cysteine residues and associated stabilizing disulphide bonds in marsupial sperm DNA. "True" sperm DNA damage is most likely associated with the KSM-3 morphotype, which shows a massive dispersion of chromatin similar to that described in other species. A model of koala sperm chromatin structure is proposed to explain the behavior of the sperm nuclei following the SCDt. Further studies are required to determine if DNA damage found in the KSM-2 morphotype is indicative of single-stranded DNA breakage, associated with an inherent lack of cysteine residues in marsupial sperm chromatin. Conversely, it will also be important to establish whether the KSM-3 morphotype is caused by an increased incidence of double-stranded DNA breakage and whether this abnormality is correlated with impaired fertility as it is in other species.
Key words: Semen Analysis •
Sperm •
DNA damage •
Koala •
in situ nick translation •
sperm chromatin dispersion test
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