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From the Departments of * Pharmacology and
Therapeutics and of Obstetrics and Gynecology,
McGill University, Montréal, Québec, Canada.
| Correspondence to: Bernard Robaire, Department of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir-William Osler, Montréal, Québec, Canada H3G 1Y6 (e-mail: bernard.robaire{at}mcgill.ca). |
| Received for publication August 14, 2006; accepted for publication September 25, 2006. |
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Abstract |
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Key words: Sperm DNA, DNA strand breaks
The demonstration that the spermatozoon can bring genetic damage into the oocyte at fertilization and contribute to the development of abnormal pregnancy outcome has led to the development of many techniques to assess sperm function and DNA integrity (Agarwal and Allamaneni, 2005; Marchetti and Wyrobek, 2005; O'Brien and Zini, 2005). Multiple assays have been developed to measure sperm chromosomal aberrations, abnormal chromatin packaging, chromatin structural integrity, and DNA breakage (for review, see Perreault et al, 2003). The chromatin structure of the sperm is very different from that of somatic cells. Indeed, during spermiogenesis, histones are replaced first by transition proteins followed by protamines (Braun, 2001), resulting in a very condensed structure of sperm DNA. While alteration of this structure or the induction of DNA strand breaks during spermatogenesis may not affect the fertilizing ability of spermatozoa, it may induce definitive changes in the genomic information transmitted to the progeny. The relative value of the tests to assess sperm integrity has been the subject of discussion due to the fact that it is not clear whether they provide overlapping or distinct information. Furthermore, our current understanding of the predictive value of these tests for abnormal reproductive outcome and effects on progeny is limited. Therefore, animal models are very useful in allowing a direct linkage between sperm chromatin integrity and effects on progeny outcome.
Previous reports from our laboratory demonstrated that subchronic exposure to the chemotherapeutic agent cyclophosphamide induced DNA strand breaks in the sperm (Codrington et al, 2004) in addition to affecting fertility by increasing preimplantation and postimplantation loss and abnormal progeny (Trasler et al, 1985). We recently developed and characterized an animal model in which male rats are exposed to the chemotherapeutic cocktail used to treat testicular cancer, BEP (Bieber et al, 2006). These animals showed decreases in reproductive organ weights (testis, epididymis, seminal vesicle, and prostate), sperm count and motility, and defects in the structure of the flagella of the spermatozoa (Bieber et al, 2006). Interestingly, these rats sired progeny which were apparently normal until the end of gestation without any change in preimplantation or postimplantation loss, but most of the pups died between birth and postnatal day 2 (Bieber et al, 2006). We hypothesize that BEP treatment induced sperm DNA damage responsible for the effect on progeny outcome; to test this hypothesis, we analyzed the chromatin integrity of sperm from these rats.
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Materials and Methods |
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Cauda Sperm Collection
Cauda epididymides were excised, trimmed free of fat, and finely minced in
PBS (1 mmol/L KH2PO4, 10 mmol/L
Na2HPO4, 137 mmol/L NaCl, 2.7 mmol/L KCl, pH 7.0) to
release spermatozoa. The spermatozoa were filtered (93 µm, SETAR, Canada)
and washed twice with hypotonic buffer (0.45% NaCl) to lyse any contaminating
cells; spermatozoa were then washed 2 times further with PBS and immediately
frozen at –80°C.
Chromomycin A3 Staining
The flow cytometry–based chromomycin A3 (CMA3) staining assay was
adapted from the slide-based method
(Bianchi et al, 1993), as
previously described (Zubkova et al,
2005). Briefly, spermatozoa were stained in CMA3 staining solution
(0.25 mg/mL in McIlvaine buffer [17 mL of 0.1 mol/L citric acid mixed with 83
mL of 0.2 mol/L Na2HPO4 and 10 mmol/L MgCl2,
pH 7.0]) for 20 minutes at 25°C in the dark, washed twice in PBS,
sonicated, and stored at 4°C in the dark until analysis. A positive
control was obtained by preincubating the spermatozoa with 200 mmol
dithiothreitol at 37°C for 10 minutes. Flow cytometry analysis was done at
the Institut de Recherche Clinique de Montréal (IRCM), using a MoFlo
High Performance Cell Sorter (DakoCytomation, Fort Collins, Colo) equipped
with a 460/10 filter and an I90 argon ion laser tuned to 457-nm line
excitation. The resulting fluorescence was detected with a 580/30 band-pass
filter and quantified using Summit v.3.1 software (DakoCytomation). A minimum
of 5000 spermatozoa per sample were analyzed.
Acridine Orange Assay
To measure the susceptibility of sperm nuclear DNA to low pH-induced
denaturation in situ, the acridine orange (AO) assay was applied, using the
method previously described as the SCSA®
(Evenson et al, 2002).
Briefly, 200-µL sperm samples (4 x 106 cells/mL in PBS)
were thawed for 2 minutes at 37°C, sonicated on ice, and mixed with 400
µL of denaturation buffer (0.08N HCL, 0.15 mol NaCl, and 0.1% Triton X-100,
pH 1.4) for 30 seconds at 4°C to denature uncondensed sperm DNA. After 30
seconds, 1.2 mL of AO staining solution (0.126 mol
Na2HPO4, 0.037 mol citric acid buffer, 1 mmol EDTA, 0.15
mol NaCL, pH 6.0 containing 6 µg/mL AO [Sigma Chemical Company, St Louis,
Mo]) was added. Exactly 3 minutes after the addition of the denaturation
buffer, spermatozoa were analyzed using a FACScan flow cytometer (BD
Biosciences, Mississauga, Canada) fitted with an argon ion laser (488-nm line
excitation). A positive control was obtained by preincubating the spermatozoa
with 20 mmol H2O2 at room temperature for 1 hour. Green
fluorescence emission of AO was measured at 515–530 nm with a band-pass
filter and red fluorescence of AO was detected through a 630–650-nm
long-pass filter. The raw data were processed using WinList Software (Verity
Software House, Topsham, Me). The DNA fragmentation index (DFI = mean red
fluorescence/total of red and green fluorescence) was analyzed according to 3
different variables, as previously described
(Evenson and Wixon, 2005): the
mean DFI, the standard deviation of DFI (SD DFI), and the percentage of cells
outside the main population (% DFI). A minimum of 5000 events were analyzed
for every sample.
TUNEL Assay
The quantity of DNA free 3'-OH ends was assessed in spermatozoa using
the TUNEL assay coupled with flow cytometric analysis using the
Apo-DirectTM kit (Said et al,
2005) with the following modifications. Frozen sperm samples were
thawed, sonicated, and resuspended overnight in 70% ethanol at
–20°Cto a concentration of 1–2 x 106
cells/mL. Samples were then centrifuged for 5 minutes at 5000 x
g, washed twice in 1 mL of wash buffer, and incubated in 100 µl
staining solution (containing the reaction buffer, terminal deoxytransferase
(TdT) enzyme, FITC-tagged deoxyuridine triphosphate nucleotides in distilled
water, according to kit instructions) in the dark at 37°C for 1 hour. The
reaction was stopped by washing twice with rinse buffer. Spermatozoa were then
resuspended in 500 µL propidium iodide (PI)/RNAse and stored in the dark
overnight at 4°C. Positive controls were obtained by pretreating the cells
with deoxyribonuclease I (100 U/µL) for 20 minutes at room temperature;
negative controls consisted of sperm incubated in the staining solution
lacking the TdT enzyme. FITC staining was analyzed using the BD FACSAria Cell
Sorting System (BD Bioscience, San Jose, Calif) fitted with a 488-nm laser.
For FITC detection, light emission was filtered through a 502-nm long-pass
filter as well as a 530/30-nm band-pass filter, while PI was detected using a
556-nm long-pass filter followed by a 575/26-nm band-pass filter. Fluorescence
was quantified by the BD FACSDiva software (BD Biosciences). A minimum of 5000
events were analyzed for every sample.
COMET Assay
DNA strand breaks in spermatozoa were evaluated using the alkaline comet
assay, as previously described (Codrington
et al, 2004). Frozen sperm samples were thawed on ice and
resuspended in PBS to a concentration of 1–3 x 105
cells/mL. Fifty microliters of the cell suspension were added to 500 µL of
molten agarose (0.5% low-melting-point grade in Mg2+ and
Ca2+ free PBS, pH 7.4, at 42°C). Fifty microliters were
immediately pipetted and evenly spread onto slides (Trevigen Inc,
Gaithersburg, Md) in duplicate, and the gel was allowed to solidify at 4°C
in the dark for 10 minutes. Slides were immersed in prechilled (4°C) lysis
buffer (2.5 mol NaCl, 100 mmol EDTA, and 10 mmol Tris-HCl; final pH 10)
containing 10% DMSO, 1% Triton X-100, and 40 mmol dithiothreitol for 1 hour on
ice, washed in distilled water for 5 minutes, and incubated for 3 hours at
37°C in lysis buffer containing 0.1 mg/mL Proteinase K. Slides were then
washed in distilled water, kept 10 minutes at 4°C, and immersed in freshly
prepared alkaline solution (1 mmol EDTA and 0.05 mol NaOH, pH 12.1) for 45
minutes in the dark. Slides were washed twice in 1X Tris-Borate-EDTA buffer
(TBE, pH 7.4) for 5 minutes, and electrophoresis was done at 14 V (0.7 V/cm)
for 10 minutes (Mini-Sub Cell GT; Bio-Rad Laboratories, Inc, Mississauga,
Canada). Slides were then fixed in ice-cold 70% ethanol for 5 minutes and
stored at room temperature. DNA was stained with 50 µL of SYBR Green
solution (Trevigen) (1:10 000 in Tris-EDTA buffer, pH 7.5) and immediately
analyzed using a DAGE-MTI CCD300-RC camera (DAGE-MTI Inc, Michigan City, Ind)
attached to an Olympus BX51 epifluorescence microscope. Fifty cells per slide
were randomly analyzed, for a total of 100 cells per animal, and fluorescent
images were scored for comet parameters. Tail length, percent tail DNA, and
tail extent moment (tail length/fraction of tail DNA) were measured using the
KOMET 5.0 image analysis system (Kinetic Imaging Ltd, Liverpool, United
Kingdom).
Statistical Analysis
Statistical analyses were done using the SigmaStat 2.03 software package
(SPSS Inc, Chicago, Ill). Significant differences due to the treatment were
determined using a 1-way analysis of variance followed by the Bonferroni test
(P < .05). Correlation analyses were done by either Pearson's
(parametric) or Spearman's (nonparametric) test, as appropriate.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Effects of BEP Treatment on DNA Strand Breaks
DNA strand breaks were measured in spermatozoa after 3, 6, or 9 weeks of
BEP treatment using 2 different assays: the TUNEL and COMET assays. Using the
TUNEL assay coupled to flow cytometry, we did not observe any effect of 3 or 6
weeks of BEP treatment. In contrast, after 9 weeks of treatment we observed a
significant dose-dependent increase in DNA strand breaks with the 2/3X and 1X
doses (Figure 3). Single and
double DNA strand breaks were also evaluated after 9 weeks of treatment using
the COMET assay; 3 parameters were analyzed. The percentage of DNA present in
the tail (% tail DNA) (Figure
4A), the tail length (Figure
4B), and the tail extent moment
(Figure 4C) were significantly
increased after the 1X dose treatment. Interestingly, spermatozoa from rats
treated with the 1/3X and 2/3X doses showed an intermediate increase, albeit
nonsignificant (Figure 4A through
C).
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Correlation Between Spermatozoal DNA Integrity Assay Parameters
In order to compare the different assays, the correlation among the
different assay parameters was evaluated. We observed that the standard
deviation of DFI (SD DFI) obtained in the AO assay correlated with the TUNEL
assay (Figure 5A), but not with
the COMET assay (Figure 5B).
The TUNEL assay was also correlated to the mean DFI (r = .405;
P = .05; n = 23), but surprisingly, this correlation did not exist
with the % DFI (r = .254; P = .24, n = 23). This lack of
correlation was probably due to the extreme variability of the % DFI (see SEM
bar in Figure 2C). We also
observed a correlation between the TUNEL assay and the COMET assay results
(Figure 5C). This finding was
expected, as these 2 assays detect DNA strand breaks in sperm DNA. The
distribution of the different doses (Figure
5C) clearly demonstrates that even if these 2 parameters are
correlated, the distribution is not well defined along the TUNEL axis, whereas
it is clear along the COMET axis. We observed that the 0x animals are tightly
grouped on the lower part of both axes and that the 1X animals always give
high COMET values. The distribution of the different rats within the 1X group
is tight on the COMET axis, while there is a wide spread on the TUNEL axis,
suggesting a lower variability with the COMET assay. Thus, the COMET assay is
more sensitive and less variable than the TUNEL assay for the extreme values;
treatment with intermediate doses may have induced intermediate levels of DNA
strand breaks that were not well detected by these techniques.
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Correlation Between Spermatozoal DNA Integrity and Spermatozoa Motility
The sperm motility from the 9 weeks 1X BEP-treated rats has been described
previously by our lab to be decreased compared to controls
(Bieber et al, 2006). We
observed that the percent sperm motility parameter (ratio of cells that are
moving at or above the minimum determined speed to total cells) was negatively
correlated with the TUNEL results (Figure
6A) and the AO assay parameters, SD DFI
(Figure 6B), mean DFI
(r = –.779; P = .022; n = 8) and % DFI (r =
–.833; P = .005; n = 8). Interestingly, conventional sperm
motility parameters (path velocity, progressive velocity, track speed, lateral
amplitude, beat frequency, straightness, and linearity) did not correlate with
the sperm DNA integrity parameters described in this paper (data not
shown).
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Discussion |
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BEP treatment altered sperm chromatin integrity only after 9 weeks of treatment with the highest dose, which is equivalent to the human treatment after adjustment for surface area (Einhorn and Donohue, 1998). Spermatogenesis is a highly time-regulated process (Clermont, 1972); 3, 6, or 9 weeks of chronic treatment reflect the effects of first exposing spermatids, spermatocytes and spermatogonia, respectively, to BEP. Our results suggest that BEP treatment induces damage in spermatogonia that cannot be repaired and is subsequently detected in epididymal spermatozoa. The nature of the damage induced in spermatogonia needs to be further investigated. Previous work suggested that differentiating spermatogonia (Meistrich, 1986) and postmeiotic germ cells (Trasler et al, 1986) are more sensitive to chemotherapeutic agents, but mitotic cells are also vulnerable (Trasler et al, 1987). Chronic treatment with the single agent cyclophosphamide resulted in a decrease in the expression of the stress-response genes in pachytene spermatocytes and round spermatids (Aguilar-Mahecha et al, 2002), but, to the best of our knowledge, changes in the gene expression profile of spermatogonia after exposure to individual or combined chemotherapeutic agents have not been reported.
In humans, the effects of BEP treatment on the DNA integrity of surviving spermatozoa have been studied using fluorescence in situ hybridization (FISH), and the results are conflicting. One study found an increase in aneuploidy (De Mas et al, 2001), another found no change (Thomas et al, 2004), and yet a third found a decrease in aneuploidy (Martin et al, 1997). The discrepancies between these 3 studies are probably due to the different clinical backgrounds and posttherapeutic delays in each patient group. Very recently, Spermon et al (2006) have assessed sperm chromatin integrity pre-BEP and post-BEP chemotherapy in humans. They observed an improvement in DNA condensation after the treatment as measured by the CMA3 assay, but the values before or after treatment remained lower than those of normal donors. In our study, we did not observe any difference in the CMA3-mean fluorescence between treated and control animals. This discrepancy may be explained by the higher compaction and lower accessibility of the sperm DNA in rat than in human (Bench et al, 1996). In addition, unlike treated patients, the animals used in our study did not have testicular cancer, nor did they undergo orchiectomy prior to the treatment. The disease itself leads to a decrease in the sperm count and abnormalities in sperm prior to the treatment (Baker et al, 2005); it is not clear whether testicular cancer also induces changes in the genomic integrity of spermatozoa. An increase in DNA strand breaks, determined by the TUNEL assay, has been described pre-BEP and post-BEP treatment in patients compared to normal donors (Spermon et al, 2006). Surprisingly, no increase has been observed when comparing the prechemotherapy and postchemotherapy sperm samples (Spermon et al, 2006), suggesting that the damage is due to the disease and not to the treatment. Nevertheless, our results strongly suggest that the drug treatment increases the number of DNA strand breaks in spermatozoa.
Semen quality tests have long been restricted to the number and motility parameters of the sperm. We observed an inverse correlation between sperm motility and DNA damage. Such an inverse correlation has been described also in thalassemic patients (Perera et al, 2002) and in men with varicocele (Smith et al, 2006). These results imply that conventional sperm parameters may be indicators of chromatin integrity and vice versa. However, BEP treatment induces oligozoospermia and a decrease in motility of the sperm (Baker et al, 2005). Further studies are needed to determine if motility and sperm chromatin integrity are linked, or if they result from distinct effects of the treatment on sperm maturation.
It is now well established that the integrity of sperm DNA and chromatin correlate with fertility (Sakkas et al, 2003). The present study suggests that the altered chromatin quality in BEP-exposed sperm will adversely impact on progeny outcome. Nevertheless, the ultimate goal of directly linking one form of sperm chromatin damage to a specific progeny outcome has not yet been achieved. In our model, BEP treatment did not affect fertility, preimplantation and postimplantation loss, or litter size on gestation day 21 (Bieber et al, 2006), suggesting that the chromatin damage observed in the sperm of these males is not involved in these processes. Nevertheless, most of the pups sired by treated males (1X BEP for 9 weeks) died before postnatal day 2 (Bieber et al, 2006). The sperm samples used in this study were collected from the animals used for the previous progeny outcome study, allowing us to make a direct correlation between these parameters. Only the TUNEL assay showed a trend of an inverse correlation with the number of surviving pups (r = –.66, P = .07, n = 8), suggesting that an increase in the number of DNA strand breaks in the sperm can lead to an increase in the death rate after birth. The use of assisted reproduction techniques, such as IVF or ICSI, allows the correlation of sperm chromatin integrity with the ability to fertilize, steps in early embryo development, and the implantation success ratio (Razavi et al, 2003; Agarwal and Allamaneni, 2004; Lewis and Aitken, 2005). Animal models may also be useful for such studies. Comparison of treatments with different effects on sperm chromatin structure and different progeny outcome defects may help to elucidate the relationship between specific sperm chromatin structure defects and progeny outcomes.
The role of sperm chromatin structure testing in routine semen analysis has been discussed (Perreault et al, 2003). The choice of assays to be included is important. In this study, we did 4 assays, giving 3 different measures of sperm chromatin integrity: its maturity and compaction, determined by the level of protamine bound to the DNA; the chromatin structure, measured by susceptibility to low-pH denaturation; and the extent of single and double DNA strand breaks, as assessed by the TUNEL and the COMET assays. While the TUNEL assay coupled with flow cytometry analysis on rat sperm was done previously (Zubkova and Robaire, 2006), it had not been compared to the COMET assay performed on slides. Using the same samples for both assays, we have shown that these 2 tests do correlate in the rat as they do in the human (Donnelly et al, 2000). Interestingly, the COMET assay seemed to be less variable and more sensitive to dose response than the TUNEL assay. We have also shown that the results of the AO assay correlate with the TUNEL assay, consistent with a recent study comparing fertile and infertile patients (Chohan et al, 2006). Surprisingly, we did not find any correlation between the AO results and the COMET assay parameters, in contrast to the findings of Aravindan et al (1997) for human spermatozoa. Differences in correlation level for the AO assay and the TUNEL or COMET assays have been described previously (Perreault et al, 2003), suggesting that each test addresses a different parameter involved in sperm chromatin integrity. Thus, the CMA3, COMET, and AO assays complement each other in predicting fertility problems; each is relevant and likely to be needed for comprehensive clinical sperm analysis.
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Acknowledgments |
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Footnotes |
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