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From the * School of Animal Studies, The
University of Queensland, Gatton, Australia;
Departamento de Biología, Unidad de
Genética, Edificio de Biología, Universidad Autónoma de
Madrid, Madrid, Spain; and the Institute of
Zoology, Zoological Society of London, Regent's Park, London, United
Kingdom
| Correspondence to: Dr Stephen Johnston, School of Animal Studies, The University of Queensland, Gatton 4343, Australia (e-mail: stevejohnston{at}uqconnect.net). |
| Received for publication May 17, 2007; accepted for publication June 29, 2007. |
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Abstract |
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Key words: DNA damage, marsupial
Postthaw mammalian sperm nuclear decondensation and instability is likely to be a prototherian/metatherian phenomenon, in that the sperm DNA in all but one of the species that have been examined (Planigale ingrami; Retief et al, 1995) appear to lack cysteine residues. The absence of this particular amino acid means that the marsupial sperm nucleus also lacks the 3-dimensional network of disulfide bonds that characteristically make eutherian sperm nuclei so stable. Consequently, marsupial sperm DNA is highly susceptible to decondensation and does so when air-dried, diluted, exposed to detergents or high concentrations of divalent ions (Cummins, 1980), and after cryopreservation (Johnston et al, 2006).
The extent to which marsupial spermatozoa exhibit nuclear decondensation after cryopreservation appears to be species dependent. For example, common wombat (Vombatus ursinus) spermatozoa, which have a similar morphology to that of koala spermatozoa (Harding and Aplin, 1990), do not show the same degree of decondensation after cryopreservation (Johnston et al, 2006) likely because koala sperm nuclei possess a large nuclear vacuole and their chromatin matrix has been shown to be more susceptible to dispersion after treatment with detergents such as Triton X-100 (Breed et al, 2001). These limited observations reinforce the importance of investigating the innate integrity of sperm DNA in the koala and any associated detrimental effects that might arise after short-term manipulation, cryopreservation, or both. Also, we need to determine whether postthaw nuclear decondensation or swelling is a result of osmotic-induced cryoinjury and whether it is coupled with single- or double-stranded DNA fragmentation.
The only systematic study of sperm DNA damage in a marsupial was that of Bennetts and Aitken (2005), who examined the effect of oxidative stress on sperm mDNA and nDNA in the Tammar wallaby (Macropus eugenii). Their study found that Tammar wallaby sperm nDNA was significantly more susceptible to oxidative damage than eutherian spermatozoa. The original sperm chromatin dispersion test (SCDt) was developed for eutherian spermatozoa (Fernández et al, 2003) and included the use of dithiothreitol (DTT) to reduce highly stabilized disulfide-bonded protamines. Because marsupial chromatin does not contain any cysteine residues, we had to refine the lysing reagents used in the SCDt protocol and to validate these results with the use of in situ nick translation (ISNT) of spermatozoa embedded in microgels. These validation studies unexpectedly revealed a constitutively high frequency of single-stranded nicks in undamaged spermatozoa and led us to propose that koala sperm DNA is normally organized in an unorthodox double-stranded configuration. Supportive evidence for this model is presented here. Sperm cryopreservation induced a further level of DNA destabilization and damage, evidenced by the increased frequency of a specific morphological subtype of spermatozoa. The establishment of a reliable technique to assess sperm DNA fragmentation in the koala will be an important tool in the further development of artificial breeding technology and the assessment of male fertility.
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Materials and Methods |
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Before the SCDt, semen straws were thawed in a water bath at 37°C, and postthaw viability was assessed immediately after thawing and after 4 hours of incubation at 37°C. Sperm viability was assessed with a supravital stain on the basis of red/green emission of 2 fluorescent dyes: acridine orange and propidium iodide (Duo-Vital kit; Chromacell SL, Madrid, Spain). Color emission differences were automatically scored with a computer-assisted semen analysis system fitted with a specific module for this purpose (SCA-Vital module; Microptic SL, Barcelona, Spain).
Semen was also collected by electroejaculation from another 3 sexually mature captive koalas for the purpose of applying the SCDt to spermatozoa that had been exposed to a range of experimental conditions. After collection, the modified SCDt was conducted on 1) undiluted semen, 2) semen that had been diluted 1:1 in TCG, and 3) spermatozoa following a standard cryopreservation procedure as documented above. Frozen koala semen straws were thawed by immersion in a 37°C water bath and evaluated immediately (T0) and after a 2-hour incubation (T2) period at 35°C. These semen samples were anticipated to provide a range of spermatozoa with various degrees of chromatin damage.
Development of the SCDt for Koala
To determine the DNA fragmentation index (DFI) in a population of koala
spermatozoa, a prototype kit (Sperm Halomax; ChromaCell SL, Madrid, Spain) was
specifically developed for the purpose. The methodology was essentially based
on the SCDt described by Fernández et al
(2003), although the absence
of disulfide bonds in marsupial sperm chromatin meant that the standard lysing
solutions and their incubation periods required modification. Two prototypes
were developed by Chromacell SL (Madrid, Spain); the primary difference
between these methodologies was whether protein depletion involved the use of
the specific protein disulfide reductor DTT. In a preliminary study, no
differences were observed between each experimental approach, so that all
subsequent experiments were performed with a lysis solution that did not
contain DTT.
Sperm samples, fresh or frozen, were diluted with TCG to achieve a final sperm concentration of approximately 10 x 106 spermatozoa/mL. Diluted sperm samples (25 µL) were added to a vial containing molten (37°C) low–melting point agarose (1%) and gently mixed. A drop of the cell suspension was then spread onto the provided Halomax pretreated slide, covered with a glass coverslip (13 x 13 mm), and placed directly onto a cold (4°C–5°C) refrigerated metallic plate for 5 minutes. Once the agar had solidified, the coverslip was carefully removed, and the preparation was then ready for further processing. Each slide was placed horizontally into 10 ml of lysing solution (pH 7.5; provided in the prototype kit) for 5 minutes. The appropriate concentration of the lysing solution and the time of incubation was a matter of trial and error. In preparation for viewing under epifluorescence microscopy, the slides were dehydrated in a sequential (2 minute) series of 70%,90%, and 100% ethanol baths and stained with a 2.5 µL/mL solution of 5x Gel Red (Biotium, Hayward, Calif). All slides were then mounted in Vectashield Mounting Medium H-1000 (Vector Laboratories, Burlingame, Calif).
DNA Fragmentation as Assessed by ISNT
After application of the SCDt, spermatozoa give rise to the differential
production of "halos" of chromatin dispersion depending on the
respective amount of DNA damage
(Fernández et al, 2005;
Enciso et al, 2006;
Gosálvez et al, 2006).
To ensure that the production of a chromatin dispersion "halo" was
correlated with the presence of fragmented DNA, ISNT was conducted. ISNT was
performed rather than the terminal transferase deoxyuridine 5-triphosphate
(dUTP) nick end labeling (TUNEL) assay because the level of DNA labeling is
typically higher with ISNT and there is limited background staining when
microgels are used.
ISNT of DNA breaks was determined on sperm samples prepared on microgel slides as for the SCDt; sperm samples were examined with and without exposure to the lysing agent provided in the prototype kit. For the latter, sperm-loaded microgels were simply dehydrated in a series of increasing alcohol concentrations as described above and then dried for 5 minutes before ISNT. For the ISNT procedure, 100 µLof reaction buffer containing 25 units of DNA-polymerase I (New England BioLabs, Ipswich, Mass) and biotin-16-dUTP in the nucleotide mix were pipetted directly onto the slide, covered with a plastic coverslip and incubated in a humidified chamber for 5, 10, 20, and 30 minutes at 37°C. After washing in Tris-Borate-EDTA buffer (pH 8), the slides were dehydrated in sequential 70%–90%–100% ethanol baths and air dried. The incorporated biotin-16-dUTP was detected after incubation for 30 minutes with avidin conjugated with fluorescein isothiocyanate (FITC). The slides were analyzed directly or, alternatively, counterstained with propidium iodide (2 µg/mL) in Vectashield (Vector, Burlingame, Calif). As a control, a designated area of the slide was incubated with the reaction buffer alone, omitting the DNA-polymerase I. The microgel between the areas with and without the polymerase was scratched off to avoid possible diffusion of the enzyme into the control area. In the case of SCDt-treated slides and to achieve the best results in polymerase activity, each slide was thoroughly washed in phosphate-buffered saline and incubated at room temperature 4 times in excess reaction buffer for DNA-polymerase I (10 mM Tris-HCl, 5 mM MgCl2, 7.5 mM DTT, pH 7.5) to remove any trace of the lysing solution, which might cause enzyme inactivation.
Digital Photomicroscope and Semiquantification of Fluorescence
Fluorescence images were captured with a Leica DMRB (Leica Microsystems,
Barcelona, Spain) microscope. Digital Images were produced as TIFF 12-bit
images with a black and white, cooled Leica DCF 300 camera with single
bandpass filters (FITC-3540B-536/617, Cy5-4040A-492/516; Semrock, Rochester,
NY). Digital image analysis was performed with the use of Leica Q-Win
software. To quantify the amount of DNA in each sperm head type, each image
was analyzed digitally after background extraction for integrated density.
Images were transformed into color by converting TIFF 12-bit images into TIFF
8-bit images with Adobe Photoshop 7.0 (Adobe Systems Incorporated, San Jose,
Calif). Color assignment to each of the 8-bit gray-level images was also
conducted with Adobe Photoshop, with the red code for DNA and the green for
proteins. This color code was selected to improve visual discrimination
between both channels, but alternative color combinations are possible.
Statistical Analysis
All data were handled with Microsoft Excel software, and statistical
comparisons were performed with StatGraphics Plus 5.1 (Academic Enterprise,
StatPoint Inc, Herndon, Va). Box-and-whisker diagrams were used for the
presentation of descriptive statistics. Nonparametric statistical tests were
chosen for the analyses because the variables departed significantly from the
normal distribution in at least one of the observations according to Shapiro
and Wilks and Kolmogorov-Smirnov tests. Accordingly, comparisons between the
different sperm morphologies (KSM-1, KSM-2, and KSM-3) were performed by
Kruskal-Wallis and Bonferroni tests with the level of significance set at
P < .05.
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Results |
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Koala Chromatin Instability as Visualized by the SCDt
When frozen-thawed koala semen samples were prepared in microgels and
exposed to the SCDt protocol, the morphology of the majority of the sperm
heads was substantially altered compared with those observed in the viability
test. Three primary morphologies were differentiated and are shown in
Figure 2. The first SCDt koala
sperm morphotype (KSM-1) corresponded to the standard rod-shaped form, as
found in the viability test (Figure
2b). The nuclei of these spermatozoa showed a homogeneous
integrated density with respect to their fluorescence, with an absence of halo
formation. The second morphotype (KSM-2) had essentially rounded nuclei with
varying degrees of diffuse halo formation
(Figure 2c to e). Although the
size of the halo varied from one cell to another, the characteristic feature
of this morphotype was one of a compacted halo of dispersed chromatin around a
dense, highly fluorescent chromatin core
(Figure 2c and e). Morphotype 3
(KSM-3) sperm nuclei were rod-shaped or round, consisting of an inner
chromatin core and a large disperse halo of stellar chromatin
(Figure 2f), corresponding to
DNA fragments diffusing from the central core.
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Application of the SCDt to Freshly Extended and Cryopreserved Koala Sperm
To explore repeatability of the koala SCDt, the proportion of koala sperm
nuclear morphotypes from the ejaculates of 3 koalas was assessed after
extension of freshly ejaculated and frozen-thawed semen samples from the same
individuals. The proportion of each morphotype after cryopreservation was
determined at hour 0 and after 2 hours of incubation at 35°C. The results
of these assessments are shown in Table
2. It should be noted that SCDt was also attempted on undiluted
semen samples, but the high protein fraction of the ejaculate made it
difficult to visualize individual spermatozoa; consequently, the SCDt of these
samples were not included in the analysis.
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Image analysis with a semiquantitative comparison of integrated density showed that each morphotype could be discriminated statistically (Figure 4). A 1-way analysis of variance revealed a difference (P < .05) between the mean integrated density of morphotypes KSM-1 and KSM-2 in both the freshly extended and frozen-thawed samples. However, the difference in the mean integrated density of each morphotype was not statistically significant when fresh and frozen-thawed samples were compared (Table 3). Given the comparatively low incidence of KSM-3, integrated density data were pooled from both extended and frozen-thawed semen samples. These observations revealed that the integrated density of KSM-3 was significantly different from that of KSM-1 and KSM-2. KSM-3 was also the only morphotype that showed integrated density values outside the main distribution; these outliers corresponded to sperm with large halos of stellar chromatin surrounded by highly degraded DNA cores.
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Discussion |
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Koala sperm nuclei that possessed a compact, rod-shaped morphology were free of labeled nucleotides, whereas slightly enlarged rod-shaped nuclei showed evidence of labeling, particularly around the periphery of the nucleus. This lack of ISNT labeling of the compact nuclei is probably associated with the inability of the polymerase to access the appropriate DNA target in a highly condensed form of chromatin. Interestingly, a similar phenomenon was also observed after protein removal following lysis associated with the use of the SCDt. Although protein removal resulted in a significant change of nuclear morphology, it also altered the DNA molecule, making it more accessible to enzyme action and ISNT so that practically all koala nuclei showed traces of DNA labeling, but to differing extents. This interesting issue needs further analysis because although the presence of hot spots for nonorthodox DNA configurations such as alkali-labile sites or apurinic or apirimidinic sites have been described in eutherian spermatozoa (Singh et al, 1989; Fernández et al, 2000; Muriel et al, 2004; Cortés-Gutiérrez et al, in press), there have been no such observations in marsupial spermatozoa. Given the absence of cysteine residues in the protamines, especially those associated with protein depletion, marsupial spermatozoa could serve as an interesting comparative model for exploring the differential presence of nonorthodox DNA conformations in spermatozoa vs somatic cell lines. We are currently exploring the hypothesis that koala sperm nuclei possess a high number of alkali-labile sites or even a nonorthodox double-stranded DNA conformation in the form of constitutive DNA nicks. This hypothesis is depicted in Figure 5 and helps to explain, to some extent, the ability of these spermatozoa to display rapid chromatin decondensation and uptake of nucleotides after ISNT, with or without protein lysis.
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Figure 5 presents a hypothetical model that helps to explain the existence of the 3 nuclear morphotypes produced after the SCDt. KSM-1 has a compacted chromatin structure and shows limited labeling to ISNT because the DNA receptive sites are protected by specific proteins. KSM-2 nuclei are characterized by swollen chromatin that has lost its structural confirmation by the removal of proteins, so that the nucleotide label has greater access to the DNA. Consequently, this results in a greater intensity of nucleotide labeling after ISNT and changed nuclear morphology (rod to rounded cell) after the SCDt. This also would explain peripheral DNA labeling observed in some nuclei after ISNT as the result of an initial destabilization of sperm chromatin structure. We propose that nucleotide uptake of both KSM-1 and KSM-2 nuclei is a result of single-stranded DNA breaks inherent to the unique composition of koala chromatin and that these breaks are unlikely to be indicative of infertility. On the other hand, KSM-3 is representative of koala sperm chromatin showing significant double-stranded DNA damage. After the removal of protein during the SCDt, small fragments of double-stranded DNA become dispersed within the microgel, resulting in receptive sites for polymerase activity and nucleotide uptake. Hence, the proportion of KSM-3 spermatozoa in the koala ejaculate after the SCDt is likely to give a more reliable estimate of lethal or sublethal sperm DNA damage.
Assuming that KSM-3 is representative of "true" sperm DNA fragmentation, the levels of DNA damage in freshly extended semen found in the 3 ejaculates of this study (2% to 12%) were similar to those described in other mammalian species (Gosálvez et al, 2006). The freshly extended sample of 1 koala in this study (K2) had a relatively high sperm DFI (sDFI) (12.2%); not surprisingly, this animal also showed corresponding poor quality seminal characteristics (Zee, personal observations).
The frequency of KSM-3 nuclei after cryopreservation was similar to that of the freshly extended semen sample, indicating that cryopreservation has little effect on sperm DNA breakage when the DNA fragmentation is assessed immediately after thawing. This indicates that the DNA molecule does not appear to suffer any appreciable breakage because of the storage methodology; a similar phenomenon has been reported in other species, including boar (Evenson et al, 1994), bull (Van der Schans et al, 2000), and humans (Duru et al, 2001). Consequently, DNA fragmentation need not be assessed immediately after collection; instead, the semen could be cryopreserved and stored for convenient evaluation in the laboratory at a later date. It is interesting that the proportion of KSM-2 nuclei postthaw decreased after 2 hours of incubation at 35°C and that this was matched with a corresponding increase in the frequency of KSM-3. This transition in the proportion of morphotypes could reflect DNA damage (KSM-3) associated with incubation at 35°C or a combination of the effects of cryopreservation and the stress of incubation. Further studies are required to examine the dynamics of sperm DNA fragmentation in freshly extended and cryopreserved semen to determine the precise nature of the cause of this damage, especially given that in vitro incubation of sperm at physiological temperatures has been shown to result in an increase of sDFI in a number of eutherian species (Ramos and Wetzels, 2001; Fraser and Strzezek, 2004; Boe-Hansen et al, 2005; Perez-Llano et al, 2006).
The koala SCDt developed in this study can be used as a powerful tool for investigating the effects of new sperm extenders or storage conditions on sperm DNA quality. In addition, postthaw modifications of koala sperm chromatin are likely to provide insights into the structure of the sperm chromatin and ultimately into the fertilization biology of this species. Further studies are required to investigate the incidence of koala sperm DNA damage at a population level, particularly in association with pathogens such as Chlamydia. Chlamydiosis is a significant problem linked with infertility in koalas (Australian and New Zealand Environmental and Conservation Council, 1998). Recent in vitro studies on human spermatozoa have demonstrated that spermatozoa incubated with Chlamydia show an increase in the amount of sperm DNA damage (Satta et al, 2006), so it will be fascinating to determine whether a similar relationship can be shown in the koala.
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Footnotes |
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References |
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