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Testis; |
Département de Biochimie, Faculté de Médecine et Sciences de la santé, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4
ABSTRACT
A precise packaging of the paternal genome during spermiogenesis is essential for fertilization and embryogenesis. Most of the nucleosomal DNA supercoiling must be eliminated in elongating spermatids (ES), and transient DNA strand breaks are observed that facilitate the process. Topoisomerases have been considered as ideal candidates for the removal of DNA supercoiling, but their catalytic activity, in the context of such a major chromatin remodeling, entails genetic risks. Using immunofluorescence, we confirmed that topoisomerase II beta (TOP2B) is the type II topoisomerase present in ES between steps 9 and 13. Interestingly, the detection of TOP2B was found coincident with detection of tyrosyl-DNA phosphodiesterase 1 (TDP1), an enzyme known to resolve topoisomerase-mediated DNA damage. The presence of gamma-H2AX (also known as H2AFX) coincident with DNA strand breakage was also confirmed at these steps and indicates that a DNA damage response is triggered. Active DNA repair in ES was demonstrated using a fluorescent in situ DNA polymerase activity assay on squash preparations of staged tubules. In the context of haploid spermatids, any unresolved double-strand breaks, resulting from a failure in the rejoining process of TOP2B, must likely rely on the error-prone nonhomologous end joining, because homologous recombination cannot proceed in the absence of a sister chromatid. Because this process is part of the normal developmental program of the spermatids, dramatic consequences for the genomic integrity of the developing male gamete may arise should any alteration in the process occur.
chromatin, DNA repair, H2AX, spermiogenesis, topoisomerase
The precise packaging of the paternal genome during spermiogenesis produces a compact and more hydrodynamic nucleus and is essential for fertilization and embryogenesis [1–3]. During the chromatin remodeling process in spermatids, the necessary removal of most somatic histones is facilitated by the incorporation of histone variants [4, 5] as well as by massive posttranslational modifications of core histones, including ubiquitination [6, 7], sumoylation [8], and hyperacetylation of histones H3 and H4 [9, 10]. During this process, most of the nucleosomal DNA supercoiling must be eliminated [11]. One hypothesis is that such a drastic change in DNA topology must require DNA strand breaks to provide the necessary swivel to relieve torsional stress [12]. Not surprisingly, we observed that all elongating spermatids (ES) between steps 9 and 12 in the mouse harbor DNA fragmentation, as shown by TUNEL. Therefore, DNA fragmentation is part of their normal differentiation program [13].
Topoisomerases have been considered as ideal candidates for the removal of DNA supercoiling in spermatids [12, 14–17]. Type II topoisomerases are involved in a variety of DNA transactions, including transcription, DNA replication, chromatid separation, and chromosome condensation [18]. Topoisomerase II alters DNA topology through a double-strand break (DSB) and its subsequent religation [18]. Two closely related isoforms have been identified in human cells: topoisomerase II
(TOP2A; 170 kDa) and topoisomerase IIβ (TOP2B; 180 kDa) [19]. Although they share high homology in their primary sequences, the two isoforms play different roles [20] and are differentially regulated during the cell cycle [21].
We and others provided evidence that a type II topoisomerase is involved in DNA fragmentation [14, 22, 23]. TUNEL labeling was greatly diminished in the presence of etoposide and suramin, two topoisomerase II inhibitors [24]. In addition, results from single-cell gel electrophoresis (COMET) of ES suggested that a majority of DNA strand breaks of normal ES were double stranded [24]. Unresolved DSBs resulting from a failure in the rejoining process by the topoisomerase II can have dramatic consequences on the genomic integrity of the developing male gamete, as these haploid cells may not rely on sister chromatids for homologous recombination-based repair. Therefore, it is crucial to investigate any evidence of impaired topoisomerase II activity and whether any signature of DNA damage response (DDR) can be found in these cells. Should this be the case, one may consider the chromatin-remodeling steps as an important source of genetic instability that may have been overlooked.
Seven-week-old male CD-1 mice were obtained from Charles River Breeding Laboratory (St-Constant, QC, Canada), maintained under standard housing conditions, and killed by CO2 asphyxiation. Animal care was in accordance with the Université de Sherbrooke Animal Care and Use Committee.
Testes were excised, decapsulated, and fixed directly in Bouin solution (Sigma-Aldrich, St. Louis, MO) at 4°C for 24–48 h and then embedded in paraffin according to standard procedures. Sections (5 µm thick) were mounted on Superfrost Plus glass slides (Fisher Scientific, St-Laurent, QC, Canada).
The rabbit polyclonal anti-
H2AX (also known as H2AFX; catalog #ab2893) and rabbit polyclonal anti-TDP1 (tyrosyl-DNA phosphodiesterase 1; catalog #ab4166) were obtained from Abcam (Cambridge, MA) and diluted 1:100 for immunocytochemistry. The rabbit polyclonal anti-TOP2B antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA; catalog #sc-13059) and diluted 1:25, whereas the anti-TOP2A antibody was obtained from TopoGEN (Port Orange, FL; catalog #2011–1) and used at a 1:100 dilution.
Rhodamine or fluorescein coupled goat anti-rabbit IgG (H + L) minimal cross-reactivity antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA; catalog nos. 111-025-045 and 111-095-045, respectively). Nonspecific binding was never observed when these secondary antibodies were incubated alone.
Antigen Retrieval and Immunofluorescence on Sections
All incubations took place in a humidified chamber. Paraffin-embedded sections were deparaffinized as previously described [13]. Heat-induced epitope retrieval (HIER) was achieved by immersing slides in a boiling solution of 10 mM Tris and 1 mM EDTA (pH 9) for 5–10 min. The slides were cooled in running tap water for 10 min and then blocked with PBS containing 1.5% BSA and 0.5% Triton X-100 for 60 min at 37°C. Primary antibodies diluted in blocking solution were incubated for 60 min at 37°C. Slides were then washed three times with PBS containing 0.1% Triton X-100 (PBST) and incubated with secondary antibodies coupled with either fluorescein or rhodamine (Jackson ImmunoResearch Laboratories) for 1 h at 37°C. Slides were washed with PBST, and DNA was counterstained with 4',6-diamidino-2-phenylindole (DAPI) or TO-PRO3 (Invitrogen Canada, Burlington, ON, Canada) and mounted with Vectashield (Vector Laboratories, Burlingame, CA).
TUNEL and Immunofluorescence on Sections
Paraffin-embedded sections were deparaffinized, and HIER was carried out as outlined above. Slides were permeabilized with blocking solution for 60 min at 37°C and incubated with TUNEL labeling mix (Roche Diagnostics, Laval, QC, Canada) containing 5 mM of CoCl2, 0.1% Triton X-100, and 0.5 nmol of dATP-Biotin (Invitrogen Canada). Four hundred units of terminal transferase as well as a 1:100 dilution of the anti-H2AX antibody were added, and the slides were incubated in a humidified chamber for 1 h at 37°C. Slides were washed with PBST and incubated in PBS containing diluted (1:100) streptavidin-FITC (Vector Laboratories) and diluted (1:200) rhodamine-coupled secondary antibodies (Jackson ImmunoResearch Laboratories) for 1 h at 37°C. Slides were washed with PBST, counterstained with DAPI, and mounted with Vectashield.
Collagenase Digestion and Squash Preparation
Testes were excised, decapsulated, and placed in a 50-ml flask containing 25 ml of freshly made 0.25 mg/ml collagenase (CLS-1; Worthington, Lakewood, NJ) in warm RPMI 1640 culture medium (Wisent, St-Bruno, QC, Canada). Testes were incubated in a shaking water bath at 32°C for 15 min or until the tubules were sufficiently dispersed. The tubules were then allowed to sediment, and the supernatant was discarded. The tubules were washed twice with 25 ml of warm RPMI.
Squash preparations were done as described by Kotaja et al. [25] with modifications. Under a transilluminating dissection microscope, 2-mm pieces of tubules were cut according to their stage-specific light-absorption pattern and transferred in 40 µl of 100 mM sucrose onto a Superfrost Plus slide. A coverslip was carefully placed over the tubule, and filter papers were used to remove excess fluid and to spread the cells out of the tubule. The slide was flash-frozen in liquid nitrogen for 20 sec, the coverslip was removed, and the cells were air-dried for 10 min at room temperature. The slides were used immediately.
In Situ Endogenous DNA Polymerase Assay
In situ endogenous DNA polymerase assays were carried out as described by Hecht and Parvinen [26] with modifications. Squash slides were incubated with TUNEL reaction mix (Roche Diagnostics) containing 5 mM of CoCl2, 0.1% Triton X-100, and 1 nmol of dUTP-FITC (Enzo Life Sciences, Inc., Farmingdale, NY) without (endogenous DNA polymerase) or with (TUNEL) 400 U of terminal transferase (Roche Diagnostics) in a humidified chamber for 2 h at 30°C. As a negative control, 5 µl of 200 mM of EDTA (pH 9) were added in the endogenous DNA polymerase mix (without terminal transferase). Slides were washed, counterstained with DAPI, and mounted with Vectashield.
Epifluorescence microscopy was done using an Olympus BX-61 motorized microscope, and confocal microscopy was performed with an Olympus FW-1000 (Olympus, Center Valley, PA).
Stage-Specific Detection of TOP2B in Mouse Spermatids
Abortive topoisomerase intermediates are known to trigger DDR in somatic cells [27]. Previous results from our group suggested that a type II topoisomerase is involved in the DNA strand breakage observed at mid-spermiogenesis steps. To confirm the nature of the topoisomerase involved, we performed immunofluorescence on Bouin-fixed, paraffin-embedded sections of mouse testes. TOP2B was present in a stage-specific manner throughout spermatogenesis (Fig. 1, A–C; Supplemental Movie 1 available online at www.biolreprod.org). It was observed in the chromocenters of Sertoli cells and in the more heavily DAPI-stained and prominent chromatin of spermatogonia. TOP2B was also observed in type B spermatogonia, as well as in some condensed chromatin from preleptotene spermatocytes to stages V–VI pachytene spermatocytes. It was absent in later pachytene spermatocytes and in round spermatids. However, it was present in nuclear foci of ES and condensing spermatids (CS) between stages IX and I, and weak labeling can be observed in the cytoplasm of CS (from stages I to VI). The flagella were also stained in late elongating and condensing spermatids (stages X to II–III). We also performed immunofluorescence for the detection of TOP2A on testis sections (Fig. 1D); spermatogonia and spermatocytes (preleptotenes to secondary spermatocytes) demonstrated strong immunoreactivity, whereas TOP2A was absent from the nuclei of all haploid cells. In ES and CS, only weak cytoplasmic staining was observed.
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The detection of the active, phosphorylated form of H2AX (H2A histone family, member X; H2AFX), H2AX, has been associated with the presence of DSBs and is an early signal for induction of the DDR in somatic cells [28–30]. We have previously demonstrated TUNEL positivity of preleptotene to zygotene spermatocytes and ES (stage IX to I), indicating that DNA strand breaks are present in these cells [13]. As the terminal transferase adds modified nucleotides to any free 3'OH DNA termini, TUNEL can not distinguish between single- and double-strand breaks. Although previous results from COMET assays indicated that a large proportion of DSBs were present in ES, we seek to establish whether the TUNEL positivity in ES was associated with H2AX as a reliable biological marker of DSBs. Strong immunoreactivity in cells initiating meiotic recombination (spermatocytes from stages VIII to I) was observed in accordance with our previously reported TUNEL positivity in these cells [13, 24] (Fig. 2, A–C; see Supplemental Movie 2, available online at www.biolreprod.org, for complete Z-stack of section of Fig. 2A; Supplemental Movie 3 available online at www.biolreprod.org). We also show that H2AX is localized to the sex bodies of pachytene spermatocytes, as this protein is known to play a role in meiotic sexual chromosome inactivation as outlined previously [31, 32]. In haploid cells, H2AX foci were clearly observed from stages IX to XII. These foci are mainly found in the heterochromatin and rarely in the chromocenter of ES and early CS. In ES, TUNEL labeling coincides perfectly with H2AX (Fig. 2D). It is noteworthy that the presence of a biological marker of double-stranded breakage also indicates that the coincident DSBs seen by TUNEL labeling do not represent an artifact resulting from the tissue preparation that would leave exposed 3'OH ends.
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TOP2B, DSBs, and Involvement of TDP1
To check for a possible impairment in TOP2B activity as one potential origin of DNA fragmentation in ES, immunofluorescence was used to detect the presence of the TDP1, an enzyme known to remove topoisomerase I and II adducts [33]. Figure 3 displays the localization of TDP1 in the seminiferous epithelium. Pachytene spermatocytes displayed a weak nuclear immunoreactivity for TDP1. At stages IX and X, TDP1 was, however, clearly found in nuclear foci distributed throughout the nucleus of ES and localized to the cytoplasm at subsequent steps. This cytoplasmic localization was found to persist in the residual bodies after spermiation at early stage IX. Because TOP2B is the sole nuclear topoisomerase detected in the nuclei of ES, the coincidence of TDP1 suggests that aborted TOP2B intermediates are indeed created, leading to DNA strand breakage.
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Endogenous DNA Polymerase Activity
Because TDP1 dislodges TOP2B cleavable complexes from DNA [34] and H2AX is activated at the site of DSB, we hypothesize that a DNA repair system may be recruited at the site of damage. During the DDR, DNA polymerase activity is required to complete the ultimate repair steps, which are the addition and replacement of intact nucleotides [35], and therefore represents an ideal marker of any active DNA repair process. Using a sensitive in situ endogenous DNA polymerase activity assay on staged squash preparations, a step-specific polymerase activity is shown in the nucleus of ES and CS (steps 9 through 13; Fig. 4, A and B). Equatorial planes of secondary spermatocytes are also labeled by the endogenous polymerase activity (Fig. 4B). Very weak activity is detected in pachytene, leptotene, and zygotene spermatocytes. As shown in Figure 4C, we observed similar results with the addition of terminal transferase. The enzymatic nature of this specific labeling is confirmed by its sensitivity to added EDTA (Fig. 4D). DNA polymerase activity in nonreplicating cells can be related only to repair; therefore, these results confirm the presence of an active DNA repair system up to step 14 of spermiogenesis.
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From the above, the step-specific detection of the proteins associated with the DSBs is reported on a spermiogenesis map (Fig. 5). As can be seen, the DDR is concentrated around the chromatin-remodeling steps defined by the detection of hyperacetylated histone H4. In the mouse, these crucial steps (9–13) last about 4 days, after which no expression of DDR factors or repair activity is detected. Hence, most of the genetic integrity of the male gamete is likely determined as spermiogenesis proceeds beyond step 13. Hence, any failure to properly repair the DNA strand breaks within this narrow window of opportunity is expected to lead to persistence of DNA damage in the mature sperm.
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Spectacular chromatin events take place during spermiogenesis as the somatic chromatin organization is replaced by a more compact, almost crystallized protaminated chromatin [1]. Transient DNA strand breaks may be formed during the process to support the overall change in DNA topology and relieve torsional stress [12]. Through their ability to generate a DSB and catalyze religation, type II topoisomerases appear to be ideal candidates to perform such tasks [14, 22–24].
We have tested anti-topoisomerase II antibodies against TOP2A and TOP2B on seminiferous tubule sections. Only the β isoform was present in foci distributed throughout the nuclei of ES (stages IX to XII), coincident with chromatin remodeling and TUNEL positivity (Fig. 5) [13]. Shaman et al. [36] found an activable TOP2B in spermatozoa, lending support to the hypothesis that it may assume a role earlier during spermiogenesis.
In the context of the condensing chromatin of ES, the capacity of TOP2B to achieve a complete catalytic cycle is questionable. The coincident phosphorylation of H2AX, a marker of DNA DSBs, supports the possibility that abortive TOP2B intermediates may be generated. Concerns were raised that the strong TUNEL labeling in ES may be a consequence of fixing the cells at a given time point, labeling the DSBs in the process of being religated by the topoII. The H2AX foci and endogenous polymerase are a clear indication that this is not the case and that a response to endogenous DNA damage is triggered. Although it was shown that H2AX is not always associated with DSBs, notably in meiotic sexual chromosome inactivation [31, 32] and in monitoring the genomic integrity independent of DNA damage [37], the coincidence of DNA DSBs by TUNEL and by H2AX foci is striking, strongly suggesting that the activation of this histone variant is triggered by the appearance of DSBs. In addition, the clear detection of DNA polymerase activity indicates that the presence of H2AX is associated with a genuine DNA damage response. Therefore, we surmise that in ES most of the DNA supercoiling is eliminated flawlessly, but in some cases a cellular signalization of DSB is activated if topoisomerase II activity is hindered. As shown in this paper, this DDR is present in all ES and seems to be part of the normal spermiogenesis program.
It is well known that abortive topoisomerase II catalysis triggers a DDR. For instance, collision between topoisomerase adducts and progressing replicative forks or transcription machinery often results in a DNA lesion [38]. In Saccharomyces cerevisae, the activity of TDP1 was first related to the hydrolysis of 3'-phosphotyrosyl bonds, which are mainly found in topoisomerase I intermediates [39–41]. However, it was demonstrated recently that the human TDP1 cleaves a broad range of substrates [42] and, in yeast, participates in topoII-mediated DNA damage [33, 34]. Thus, one can hypothesize that TDP1 actively removes TOP2B cleavable complexes during spermiogenesis, leaving a DSB. TDP1 was first identified by mass spectrometry following co-immunoprecipitation of hyperacetylated histone H4 (AcH4) from sonication-resistant spermatid extracts (Joly and Boissonneault, unpublished data). The association of a repair enzyme such as TDP1 with AcH4 is consistent with the known requirement of histone hyperacetylation at damage sites [43]. In this report, the presence of TDP1 in ES was confirmed by immunofluorescence of testis sections. Interestingly, the step-specific appearance of TDP1 during spermiogenesis is coincident with the presence of TOP2B in the nucleus of ES. The presence of TDP1 makes it likely that unprocessed DNA ends from the TOP2B catalysis may be responsible for the formation of DSBs.
DNA Repair System as Part of Normal Spermiogenesis
The presence of H2AX foci in ES and early CS (stages IX to I) supports previous observations that DNA fragmentation consists of DSBs and provides more insights about this critical step. The presence of H2AX and endogenous DNA polymerase activity is a clear indication that DDR signaling is taking place during chromatin remodeling in spermatids. Our observation is in agreement with previous work by Meyer-Ficca et al. demonstrating the presence of H2AX as well as poly(ADP-ribosyl)ation in rat spermiogenesis [44], though, in the latter case, the coincidence with DNA strand breakage was not established. Consistent with these observations, the knockout of the Parp2 gene in mice was associated with severely compromised differentiation of spermatids and delays in elongation [45].
We found endogenous DNA polymerase activity present in ES and CS, whereas much weaker activity was detected in premeiotic and meiotic cells, with the exception of equatorial planes of secondary spermatocytes. The presence of endogenous DNA polymerase in equatorial planes of secondary spermatocytes may also be related to repair of topoisomerase adducts because the enzyme is required for the segregation of sister chromatids during meiosis I [18]. These results are in accordance with those of Hecht and Parvinen [26], although in their case the labeling was not mainly associated with ES. The presence of DNA polymerase activity up to the CS steps indicates that DNA repair may still proceed to completion despite the major chromatin restructuring in these cells and may explain why DNA strand breaks are normally no longer found in steps 15–16 spermatids even when nuclear decondensation is performed [13].
Given the haploid character of spermatids, one can assume that they must rely on a process related to nonhomologous end joining (NHEJ) for DNA repair; therefore, it is not surprising that components of NHEJ have been previously associated with spermatids [46–48]. However, processes related to NHEJ are recognized as being error-prone, so spermatids may represent a major source of genetic instability. Unresolved paternal DSBs that have persisted in the mature sperm, leading to high DNA fragmentation level, may not be processed due to the limited repair capacity of the oocyte [49, 50]. In addition, completed NHEJ repair process in these haploid cells may induce mutations that will not be corrected in the zygote and will, therefore, be transmitted to the offspring. Special attention in this regard should be given to the Y chromosome because abnormalities of this chromosome are the second most common cytogenetic anomalies observed in infertile men; micro or partial de novo deletions of the long arm (Yq) have been observed in around 3% of infertile men [51]. The Y chromosome is very vulnerable to DNA deletions because of its inability to participate in recombination repair [52]. Further evidence is needed to establish the consequence of the endogenous DNA strand breaks in fertilization and embryo development.
Our results are consistent with other evidence from the literature, allowing us to propose what must be considered as an early model of the events taking place during steps 9 to 13 of mouse spermiogenesis (Fig. 6). The hyperacetylation of the histones H3 and H4, as well as other posttranslational modifications, weakens the affinity of histones to DNA. Somatic nucleosomes are partially removed from DNA, leaving chromatin with free supercoils, which are substrates of TOP2B that relax DNA for the proper deposition of transition proteins and protamines. In this process, part of the TOP2B pool may get trapped in a cleavable complex or may have a partial activity, so TDP1 would be needed. DSBs are left by the cleavage of TDP1 from the DNA-topoisomerase complex, which activates H2AX through the phosphorylation of a member of the phospho-inositide 3-kinase family [28] and triggers the addition by PARP2 of poly(ADP-ribosyl) polymer on surrounding histones [44, 45]. At this point, we hypothesize that elements of the NHEJ are recruited to the DSB. Free DNA ends are then processed, and gaps are filled by a DNA polymerase and ligated. Transition proteins and protamines may be involved in the repair process, as they are known to facilitate DNA ligation both in vitro and in cellulo [53].
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Relationship to Human Fertility
The origin of DNA fragmentation in human spermatozoa remains unknown, but multiple sources have been proposed, including abortive apoptosis, abnormal chromatin packaging, and generation of reactive oxygen species [1, 54, 55]. The requirement of a DNA repair system in the normal differentiation program of spermatids supports the concept that transient DNA fragmentation observed during spermatid chromatin remodeling may persist in infertile human spermatozoa because of impairment in the repair process. Unresolved DSBs resulting from a failure in the rejoining process can have dramatic consequences on the genomic integrity of the developing male gamete. Not surprisingly, increasing evidence from the literature points to alterations in the nuclear integrity of the male gametes as the cause of de novo genetic disorders, developmental and morphological defects, cancer, and miscarriage [56–59]. Furthermore, sperm DNA fragmentation can also compromise assisted reproductive technology (ART) [59].
In conclusion, we provided evidence of a DNA repair system in normal mammalian spermiogenesis likely induced in response to endogenous DNA strand breaks. Given the haploid character of spermatids, an error-prone repair system such as NHEJ must operate. These findings lend strong support to the view that the origin of DNA fragmentation in sperm may lie within the chromatin-remodeling steps in spermatids.
ACKNOWLEDGMENTS
The authors wish to thank Dr. Leonid Volkov for his technical advice regarding confocal microscopy and Leila Jaouad for her technical assistance.
FOOTNOTES
1Supported by the Canadian Institutes of Health Research (grant MOP-74500) to G.B. Part of this work was presented at the 53rd annual meeting of the Canadian Fertility and Andrology Society in Halifax, Nova Scotia, Canada, September 26–29, 2007. 
Correspondence: 2Guylain Boissonneault, Département de Biochimie, Faculté de Médicine et Sciences de la santé, Université de Sherbrooke, 3001 12ième Ave Nord, Sherbrooke, Québec, Canada J1H 5N4. FAX: 819 564 5340; e-mail: guylain.boissonneault{at}usherbrooke.ca
Received: 11 July 2007.
First decision: 7 August 2007.
Accepted: 5 November 2007.
REFERENCES
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  M. L. Meyer-Ficca, J. Lonchar, C. Credidio, M. Ihara, Y. Li, Z.-Q. Wang, and R. G. Meyer Disruption of Poly(ADP-Ribose) Homeostasis Affects Spermiogenesis and Sperm Chromatin Integrity in Mice Biol Reprod, July 1, 2009; 81(1): 46 - 55. [Abstract] [Full Text] [PDF]
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