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Department of Biochemistry, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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acetylation, chromatin remodeling, developmental biology, DNA strand breaks, gamete biology, gamete integrity, gametogenesis, germ cells, spermatid, spermatogenesis, spermiogenesis, TUNEL
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Any alteration in the protein exchange process leading to chromatin remodeling may therefore lead to dramatic consequences on the genetic integrity of the male gamete, as the condensing spermatids may not possess the proper machinery for elaborated DNA repair. For instance, deletion of either of the two major spermatidal transition proteins, TNP1 and TNP2, produces alterations of the condensation state of the epididymal sperm as well as an increase in DNA susceptibility to denaturation [10, 11], while an increase in TUNEL positivity in the developing spermatids occurred in a double null mutant of these genes [12]. The fertilizing potential and early embryonic development was impaired in these double knockout mice possibly because of the underlying alteration in genetic integrity [13, 14]. Similarly, it was recently demonstrated that sperm with protamine 1 and protamine 2 deficiency harbors DNA damage as shown both by the comet assay [15] and by fragment release in agarose gel electrophoresis [16]. These sperm also fail to produce viable embryos. Hence, a proper nucleoprotein exchange during spermiogenesis is apparently required to avoid persistence of DNA strand breakage in the mature sperm.
Interestingly, the endogenous DNA strand breaks generated during the chromatin remodeling steps in both mouse and human were found to be perfectly coincident with the presence of hyperacetylated H4, suggesting a close relationship between the formation of DNA strand breaks and the acetylation state of the histones. The mechanism at the origin of the formation and repair of strand breaks is yet unknown but deserves to be investigated, as it may be vulnerable. Any impairment in the process may alter DNA integrity, leading to mutations and genetic disorders of the newborn.
Previous studies demonstrated the presence of topoisomerase II in elongating spermatids of both rats and chicken [17–19]. Topoisomerase II catalyzes different DNA isomerization reactions by a double-strand breakage and rejoining mechanism that is dependent on ATP hydrolysis. However, it remains unclear whether topoisomerase II is the unique enzyme responsible for the production of strand breaks. Involvement of such a type II topoisomerase activity would therefore suggest the occurrence of transient double-stranded breaks. Given the haploid character of elongating spermatids and their limited DNA repair capacity [20], transient double-strand breakage generated by abortive reaction intermediates of the topoisomerase II catalysis can potentially be harmful. It is therefore of prime importance to characterize the nature of the transient DNA strand breakage in elongating spermatids and to identify the topological enzymes operating in that cellular context. In an effort toward this goal and using the mouse model, we present evidence that double-stranded DNA breaks are indeed generated during the chromatin-remodeling process in elongating spermatids making use of the single-cell electrophoresis (comet assay). In addition, we demonstrate that histone acetylation is required for the formation of DNA strand breaks and that a type II topoisomerase is involved.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Trichostatin A (TSA) and suramin were both purchased from Sigma (St. Louis, MO). Topotecan (Hycamtin) and etoposide (VePesid) were obtained from GlaxoSmithKline (Philadelphia, PA) and Bristol-Myers Squibb (New York, NY).
Animals and Cell Preparation
Seven-wk-old male CD-1 mice were obtained from Charles River Breeding Laboratory (St-Constant, QC, Canada), maintained under standard housing conditions, and killed by cervical dislocation. Animal care was in accordance with the Canadian Institutes of Health Research guidelines and the University of Sherbrooke Animal Care and Use Committee. Decapsulated testes were minced with a scalpel and the cells resuspended in 1.5 ml of PBS and filtered through a 60-µm Nitex mesh (G and K Products, Dollars Des Ormeaux, Canada).
Acridine Orange Staining and Cell Sorting
Mouse testes were frozen in liquid nitrogen and thawed at 37°C. Cell suspensions were prepared as described previously. A two-step acridine orange (AO) staining was performed on the filtered cell suspensions as described for the sperm chromatin structure assay (SCSA) [21]. Fluorescence-activated cell sorting of the AO-stained cells was performed using a fluorescence-activated cell sorting (FACS) Vantage flow cytometer and the CellQuest software (Becton Dickinson, San Jose, CA). Before each sample analysis, the instrument was calibrated to optimize alignment and sensitivity with FITC and PE-fluorescent CaliBRITE beads (Becton-Dickinson, Mississauga, ON, Canada). Following ion-laser excitation at 488 nm, emission was recorded using 530/30 nm (FL1) and 630/22 nm (FL3) optical filters. The forward and side scatter was determined using a 488-nm argon-ion laser. Samples were gated on forward and side scatter to exclude debris. Cells were sorted with PBS as sheath buffer. Some cells were immediately resubmitted to AO staining, centrifuged, and applied onto glass slide for examination of both the chromatin condensation state and purity by fluorescence microscopy.
Hyperacetylated Histone H4 Immunofluorescence and TUNEL Assay
Cell suspensions were smeared on a microscope slide previously coated with poly-L-lysine 0.01% and fixed with 2% formaldehyde for 10 min at room temperature. After three washes in deionized water, the cells were prepared for immunofluorescence as previously described [22]. Briefly, fixed cells were treated with 0.1 mg/ml RNase (Calbiochem-Novabiochem Corp., La Jolla, CA) for 1 h at 37°C. They were then incubated three times 30 min at room temperature in PBST buffer (0.15% Na2HPO4, 0.024% KH2PO4, 0.8% NaCl, 0.02% KCl, 0.1% Triton X-100, pH 7.4) containing 1.5% BSA as a blocking solution. The cells were then incubated overnight in the presence of a polyclonal antibody against hyperacetylated histone H4 (Upstate Biotechnology, Lake Placid, NY) at a dilution of 1:100 in PBST buffer containing 0.13% sodium azide at room temperature. After three washes in PBST at room temperature, the cells were incubated in blocking solution containing a rhodamine-conjugated, goat anti-rabbit IgG secondary antibody (BIO/CAN Scientific, Mississauga, ON, Canada) at a final dilution of 1:200 and for 2 h at room temperature. As a control for nonspecific binding, the slides were processed as described previously, except that the primary antibody was omitted. A sensitive TUNEL assay using fluorescence was performed immediately after the immunofluorescence as briefly outlined. After three washes in PBS, the slides were preincubated inside a humidified chamber with TdT buffer (Roche Diagnostics, Laval, QC, Canada) for 30 min at 37°C. The TdT buffer was discarded and replaced by 50 µl of fresh buffer containing 25 U of terminal transferase and 0.5 nmol of biotin-16-dUTP (Roche Diagnostics). The reaction was allowed to proceed for 60 min at 37°C and stopped by washing the slides twice in PBS, then with TN buffer (30 mM Tris-HCl, 300 mM NaCl, pH 7.5) containing 0.1% Triton X-100. The slides were then incubated with Fluorescein-Avidin DN (Vector Laboratories, Burlingame, CA) diluted 1:100 in AP buffer (50 mM Tris-Cl, 150 mM NaCl, pH 7.5) containing 0.1% Triton X-100 for 60 min at room temperature to reveal the biotinylated UTP extension of the free 3'-OH ends. The slides were then washed twice in AP buffer containing 0.1% Triton X-100 and finally mounted with coverslips using Vectashield mounting medium containing DAPI. As a control for nonspecific labeling, the TUNEL reaction was carried out without the TdT enzyme. The cells were then counterstained and mounted with coverslips using Vectashield mounting medium containing 4',6'-diaminido-2-phenylindole (DAPI) (Vector Laboratories). The fluorescence was examined under a Zeiss Axioscop 2 microscope equipped with a 40x objective and a Spot cooled color digital camera (Diagnostic Instruments Inc., St. Sterling Heights, MI). Step-specific labeling was determined based on the nuclear morphology of the elongating spermatids.
Comet Assay
Single-cell electrophoresis (comet assay) was performed as described initially by Singh et al. [23] with modifications. Briefly, FACS purified spermatids were mixed with 1% low-melting-point agarose and deposited onto a frosted microscope slide (Erie Scientific, Portsmouth, NH) previously coated with 1% agarose. The slides were incubated in lysis buffer (2.5 M NaCl, 10 mM Tris-HCl, 100 mM EDTA pH 10, 1% Triton X-100, 10 mM DTT) at 37°C for 1 h. Proteinase K was then added to this solution to a final concentration of 0.01 mg/ml, and the lysis was allowed to proceed O/N at 37°C. After three washes in water, the slides were immersed in either a neutral (TAE 1x, pH 8.0) or an alkaline (TAE 1x, pH 12.4) solution for 40 min at 4°C as indicated. The slides were then submitted to electrophoresis at 0.7 V/cm for 20 or 10 min for neutral and alkaline conditions, respectively. After three washes, the slides were stained by SYBR gold (Molecular Probes, Eugene, OR) and visualized by fluorescence microscopy as described previously. Three slides per sample were made, and pictures of 50 comets, selected randomly, were taken from each slide. The selected comets were analyzed using the CASP freeware to assess the fraction of DNA in the tail [24].
Topoisomerase II and HDAC Inhibitors in Short-Term Culture of Testicular Cells
Mouse testis cells suspensions were prepared as described previously. Cells were incubated at 35°C in 10 ml of DMEM culture media containing 10% calf serum, ampicillin, and the topoisomerase inhibitor as indicated. All drug treatments were carried out for 4 h except for suramin, which was applied for a period of 24 h. Suramin was used at a final concentration of 50 µg/ml (35 µM), whereas etoposide was used at a final concentration of 1 µM. The histone deacetylase inhibitor TSA was used at a final concentration of 1 µg/ml. The cells were then harvested by centrifugation, washed twice with PBS, and deposited onto microscope slides for immunofluorescence and TUNEL assay as described previously.
In Vitro Activity of Topoisomerase II Inhibitors
For the preparation of the nuclear extract from purified spermatids, all operations were carried out at 4°C. One milliliter of lysis buffer (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT, and 0.5 mM PMSF) was added to the pelleted cells. The cells suspension was kept on ice for 15 min, and 62.5 µl of NP-40 10% were then added and the cells agitated for 10 sec. The cell lysate was incubated for 5 min, centrifuged at 18 000 x g for 1 min, and then resuspended with 100 µl of extraction buffer (20 mM HEPES pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 1 mM PMSF). The extraction was allowed to proceed on a rotating wheel for 60 min at 4°C. The nuclear extract was then centrifuged for 5 min at 13 000 rpm at 4°C, and the supernatant was transferred into a new tube and stored at –20°C. The protein concentration was determined using the DC reagent (Bio-Rad, Hercules, CA).
Topoisomerase II inhibition reaction was performed at 37°C for 30 min with 200 ng of kDNA (TopoGEN, Columbus, OH) in a final volume of 20 µl of decatenation buffer (50 mM Tris pH 7.5, 100 mM KCl, 10 mM MgCl2, 0.5 mM ATP, 0.5 mM DTT, 30 mg/ml BSA, 1 mM EDTA). Six micrograms of nuclear extract, prepared as described previously, were added to each decatenation reaction. Where indicated, suramin or etoposide was added at a final concentration of 50 or 100 µM, respectively. The reactions were stopped by adding 2 µl of loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF, 30% glycerol). The samples were resolved in a 0.8% agarose gel prepared in TBE buffer (90 mM Tris-Borate, 90 mM boric acid, 2 mM EDTA pH 8.0). The migration was allowed to proceed for 2 h and the gel stained by ethidium bromide. As a positive control, 200 ng of kDNA were decatenated using 2 U of a commercial preparation of topoisomerase II (TopoGEN) as described previously.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The successful purification of mouse elongating spermatids, representing steps 9–12 of spermiogenesis, allowed us to proceed for the determination of the nature of DNA strand breaks in these cells. To this aim, round, elongating and elongated spermatids were submitted to single-cell electrophoresis assay (or comet) in both alkaline and neutral conditions. In this assay, DNA fragmentation generates a comet-like tail behind individual nucleus after their electrophoresis in a thin layer of agarose covering a microscope slide. Results of the comet assays are summarized in Figure 2. Both round and elongated spermatids displayed no detectable DNA strand breakage based on the TUNEL analysis of testis sections that we described previously [9] and were used as baseline. In neutral conditions, only double-stranded breaks contribute to the tail formation. The percentage of DNA in the tail was found to be significantly higher in elongating spermatids reaching 26.48 ± 8.22% compared to 6.65 ± 1.16% and 6.03 ± 0.94% for round and elongated spermatids, respectively (Fig. 2A). This result suggests that double-stranded breaks are present in elongating spermatids. The comet assay was then carried out in alkaline conditions. In such conditions, both double- and single-stranded breaks are detected. As expected, the percentage of DNA in the tails was also much greater in elongating spermatids, reaching 48.4 ± 17.6%, compared to 14.4 ± 3.3% and 12.3 ± 4.4% in round and elongated spermatids, respectively (Fig. 2B). Because alkaline and neutral electrophoresis must be carried out in different conditions, the extent of tail formation must be expressed relative to the baseline level found in round and elongated spermatids for each condition. When such normalization is performed, the extent of DNA strand breakage in alkaline and neutral conditions can then be compared. Hence, the relative increase found in elongating spermatids in alkaline conditions is not greater than the increase found in neutral conditions (Fig. 2C). These results therefore indicate that most of the DNA strand breaks in elongating spermatids are of double-stranded type. Typical comets generated from round, elongating and elongated spermatids in both alkaline and neutral conditions are shown in Figure 2, D–I.
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The perfect temporal and nuclear coincidence of H4 hyperacetylation (AcH4) and DNA strand breaks in elongating spermatids suggested that a close relationship may exist between this important posttranslational modification of histones and the generation of DNA strand breaks [9]. Short-term cultures of testicular cells were incubated with or without TSA, a potent inhibitor of HDAC activity, as described under Material and Methods. Isolated cells were staged based on morphology and hyperacetylated H4 immunoreactivity. As shown in Figure 3A, the typical immunoreactivity against the hyperacetylated form of H4 is detected from steps 8–9 to steps 11–12. No fluorescence was detected if the primary antibody was omitted (data not shown). The coincidence of TUNEL positivity at these steps is also demonstrated by the green fluorescence. Omission of the terminal transferase enzyme in the TUNEL assay resulted in the complete disappearance of the green fluorescence (data not shown). The treatment of cells with 1 µg/ml of TSA resulted in a general increase in AcH4 immunoreactivity and TUNEL positivity between steps 9 and 12. In addition, spermatids from steps 1–8 were now immunoreactive for AcH4 in agreement with the results previously reported by Hazzouri et al. [22] (Fig. 3B). Interestingly, these round spermatids also became TUNEL positive following TSA treatment. These observations therefore suggest that histone hyperacetylation, resulting from the inhibition of HDAC, is sufficient to promote formation of DNA strand breaks in the round spermatids.
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Topoisomerase II has been demonstrated to be present in elongating spermatids of both chicken and rat [17–19]. However, it was not clear whether it was the unique enzyme involved in the strand breakage and ligation observed or if other topoisomerases or endonucleases may be present. To resolve this important issue, a short-term culture of spermatids was incubated with two topoisomerase II inhibitors having different mechanisms of action. Etoposide inhibits topoisomerase II activity by stopping the reaction at the complex stage, that is, when the covalent bonds between DNA and topoisomerase II are formed [26], whereas suramin inhibits topoisomerase II by preventing its phosphorylation and binding to DNA [27, 28]. The inhibitory activity of both drugs has been confirmed using an in vitro decatenation assay with nuclear extracts of purified spermatids. As shown in Figure 4A, left panel, the catenated kinetoplast substrate remains trapped in the well (lane 2), while decatenation, induced by the enzyme in the nuclear extract, produces a short ladder of topoisomers with complete decatenation seen as the fastest migrating band (2.5-kb minicircle, lane 3). Addition of suramin produces a near complete inhibition of decatenation (lane 4), whereas a complete inhibition was observed with etoposide (lane 5). The activity of a commercial preparation of topoisomerase II, generating a complete decatenation of the 2.5-kb minicircle, is shown for reference (Fig. 4A, right panel). Neither drug altered the DNA relaxation activity of topoisomerase I (not shown). Since these inhibitors do not produce accessible 3'OH ends, one can therefore expect that, if topoisomerase II is the sole enzyme responsible for the transient strand breakage and ligation, the use of either of these drug in short-term cultures of testicular cells will result in a reduction or elimination of the TUNEL positivity normally observed between steps 9 and 11. Figure 4, B–D, demonstrates that this is indeed the case. Incubation of spermatids in the presence of etoposide completely abrogated the TUNEL positivity between steps 9 and 12 (Fig. 4C) without altering H4 hyperacetylation. Suramin also severely decreased the TUNEL positivity, as only residual green fluorescence is detected at steps 9–10 (Fig. 4D). These results therefore indicate that topoisomerase II alone is involved in the generation of the strand breakage and ligation process. This observation is also in agreement with previous results from the comet assays, demonstrating that double-stranded breaks, typical of topoisomerase II catalysis, are predominant. Interestingly, the TSA-induced TUNEL positivity of round spermatids or enhanced positivity in elongating spermatids is also not detected in the presence of etoposide. This suggests that topoisomerase II is also involved in the formation of de novo DNA strand breaks when stimulated by TSA (Fig. 4E).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present report, two potent topoisomerase II inhibitors, added to short-term cultures of spermatids, prevented the detection of DNA strand breaks. Although they are known to operate via a different mechanism, we have shown that both were able to prevent the in vitro decatenation of kinetoplast DNA. Topoisomerase II catalyzes the double-stranded cleavage of DNA, allowing passage of a second DNA duplex through the break, therefore changing the linking number in steps of two. The generation of transient double-stranded breaks by this enzyme is in accordance with the detection of double-stranded breaks by the comet assay. The generation of double-stranded breaks by the topoisomerase II reaction has several implications regarding the potential genetic consequences of any impairment in that process. In the spermatids, abortive topoisomerase II intermediates could result in the exposure of double-strand breaks, leading to genetic instability. Homologous recombination do not operate in the haploid-condensing spermatids because no homologous chromosome or sister chromatid is available. Although the DNA repair capacity of elongating spermatids has been questioned [20], the presence of alternative, more error-prone DNA repair mechanisms occurring at these steps is now gaining wider acceptance [34]. The mutagenic potential of the transient strand breakage at midspermiogenesis steps may be exemplified by the recent demonstration that gap-filling synthesis could be responsible for trinucleotide expansion repeats within specific genes [35].
Any failure to eliminate the unconstrained DNA supercoils by topoisomerase II can alter the DNA condensation process, so the high level of DNA compaction of the sperm DNA may not be achieved. Over the past 10 yr, a clear correlation has been established between the presence of sperm DNA strand breaks and the altered condensation state of chromatin [36–39]. This altered state of sperm chromatin also correlates with decreased fertility and failure of embryo implantation [5, 40–42]. It seems therefore logical to consider that impairment in the factors leading to DNA topological transitions taking place in elongating spermatids may be among the leading causes of the perturbed DNA condensation state in the mature sperm.
The possible involvement of histone acetylation in promoting DNA strand breakage has been suggested from our previous observation that the detection of hyperacetylated H4 in mouse elongating spermatids is coincident with TUNEL positivity detected from steps 9–12 [9]. As shown in the present study, altering the HAT/HDAC balance with TSA led to the de novo appearance of hyperacetylated H4 in spermatids, confirming the previous observation made by Hazzouri et al. and suggesting that histone deacetylase activity is present within these cells [22]. TSA also led to the appearance of DNA strand breakage in round spermatids, clearly suggesting that a functional relationship exists between histone hyperacetylation and the production of DNA strand breaks. This also implies that the enzyme involved in the generation of strand breaks is present in the round spermatids but may not access DNA in the normal situation. Since etoposide also prevented the TSA-induced DNA strand breakage in round spermatids, topoisomerase II therefore also appears to be the enzyme responsible for the induced TUNEL positivity in these cells. The association of H4 hyperacetylation and enhanced sensitivity to endonucleases has been suggested previously [43], and the role of histone H4 acetylation in double-strand break repair has been demonstrated in yeast [44]. It is therefore not surprising that chromatin remodeling in spermatids has been shown to be highly sensitive to the action of exogenous DNase I, indicating that a transient state of increased accessibility is generated [29]. At this point, one may only speculate that such a transient state of H4 hyperacetylation may promote the repair of the double-stranded breaks should any abortive intermediate of the topoisomerase II catalytic activity arise. Interestingly, a decrease in H4 acetylation has been associated with an alteration in the histone-to-protamine exchange in infertile men [45, 46], emphasizing the possibility that histone modification plays a crucial role in the developmental integrity of male gamete. In accordance with this concept, the human CDY and mouse Cdyl gene products recently characterized possess HAT activity, and their expression pattern is consistent with a role in mediating H4 hyperacetylation at midspermiogenesis steps [47]. The CDY genes map within AZFc, a region of the Y chromosome frequently deleted in infertile men suffering from spermatogenic failure [48].
In the present paper, we have used the comet assay to demonstrate the double-stranded nature of the transient DNA strand breaks at midspermiogenesis steps. We have demonstrated that these strand breaks are eliminated on incubation of spermatids with specific inhibitors of topoisomerase II. Hence, no other enzymes such as endogenous nucleases or type I topoisomerases appear responsible for the generation of DNA strand breaks. One can therefore hypothesize that the transient state of histone hyperacetylation during the chromatin remodeling in elongating spermatids allows topoisomerase II to gain access to DNA to relieve supercoiling. Hyperacetylation occurs as a result of an increase in the HAT/HDAC ratio such as following TSA treatment. In this condition, de novo DNA double-stranded breaks can be produced by the activity of topoisomerase II. Further investigations will be needed to establish the link between the proper control of histone acetylation and genetic integrity of the male gamete.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Guylain Boissonneault, Département de Biochimie, Faculté de Médecine, Université de Sherbrooke, 3001 12ième Ave Nord, Sherbrooke, Québec J1H 5N4, Canada. FAX: 819 564 5340; guylain.boissonneault{at}usherbrooke.ca
Received: 5 October 2004.
First decision: 16 November 2004.
Accepted: 9 March 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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