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Research Article |
Institute for Biogenesis Research, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii 96822
ABSTRACT
We previously demonstrated that mammalian spermatozoa contain a nuclease activity that cleaves DNA into loop-sized fragments. We show here that this activity is mediated by a nuclear matrix-associated topoisomerase IIB (TOP2B) interacting with an extracellular Mn2+/Ca2+-dependent nuclease. Together, these enzymes cleave all of the DNA into fragments of 50 kb, and this cleavage can be reversed by EDTA. If dithiothreitol is included, the nuclease digests the DNA, and if the protamines are removed the DNA is completely digested. A similar, TOP2B-mediated, chromatin fragmentation, which is reversible, followed by digestion of the DNA by an intracellular nuclease occurs in somatic cells during apoptosis. The extracellular location of the sperm nuclease made it possible to reconstitute the fragmentation activity in isolated spermatozoa, thus allowing us to identify two novel aspects of the mechanism. First, the fragmentation of all of the DNA to 50 kb by TOP2B required the addition of the extracellular nuclease or factor. Second, the subsequent, complete digestion of the DNA by the nuclease could be inhibited by etoposide, suggesting that the nuclease digestion requires TOP2B religation of the cleaved DNA. These data are the first demonstration of an active TOP2B in spermatozoa, suggesting this inert chromatin may be more active than previously thought. They also show that the unique chromatin structure of spermatozoa may provide an important model to study the regulated degradation of chromatin by TOP2B and associated nucleases.
apoptosis, epididymis, gamete biology, sperm, testis
INTRODUCTION
During mammalian spermiogenesis virtually all the histones are replaced with protamines [1], organizing the DNA into tightly packed toroids [2, 3]. Each toroid contains up to 60 kb of DNA [4], which is resistant to DNAse I digestion [5]. We have shown that this highly compact sperm DNA, like somatic cell chromatin, is organized into loop domains roughly 50 kb in length that are attached at their bases to a proteinaceous nuclear matrix [6–8]. We have recently provided experimental evidence to support our model that each protamine toroid is a single DNA loop domain [5] (Fig. 1). Our data suggest that the protamine toroids are connected by DNAse I-sensitive chromatin segments that we have termed toroid-linker regions.
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More recently, we demonstrated that spermatozoa from three different species contain nuclease activities that digest DNA to loop-sized fragments of approximately 50 kb [9]. Similar nucleolytic activities have been shown in somatic cells during apoptosis. Specifically, the early stages of apoptosis are accompanied by activating topoisomerase II-mediated cleavage of DNA into high-molecular weight fragments that are roughly the size of a single DNA loop domain [10]. TOP2B also has been shown to interact with the caspase-activated DNAse, DFFB, which can induce apoptotic chromatin condensation [11]. The nuclease activity in spermatozoa resembles that of the early stages of apoptosis in somatic cells, because they both result in 50-kb fragments. However, we had no evidence that TOP2B was involved, nor for an associated nuclease that could further digest the DNA into much smaller segments, as in somatic cell apoptosis.
TOP2B is a large, homodimeric enzyme that allows for the controlled nicking, relaxation, and religation of DNA where chromatin topology must be adjusted for strain, torsion, or concatenation and is required for DNA replication and chromosome segregation [12]. TOP2B causes transient, double-stranded DNA breaks as part of its mechanism, and during apoptosis in somatic cells these breaks are not re-ligated. It is located at the bases of DNA loop domains in somatic cells [13, 14] and is one of the major scaffolding proteins of mitotic chromosomes and interphase nuclear matrices [15–17]. TOP2B likely is required for the transition from histone-bound to protamine-bound chromatin during spermiogenesis [18]. However, the presence of TOP2B in mature spermatozoa is controversial [19, 20]. Several investigators have suggested that the DNA nicks seen in mature sperm cells are probably not the product of apoptosis, but rather are the result of topoisomerase-mediated breaks that occur during spermatogenesis that are not repaired [21, 22].
In this report we tested the hypothesis that the mature mammalian spermatozoa contains an active TOP2B, and that this enzyme mediates apoptotic-like cleavage in concert with a nuclease that digests the sperm DNA.
MATERIALS AND METHODS
Animals and Cell Preparation
Male BD2F1 mice from Charles River Laboratories or the National Cancer Institute were maintained under standard housing conditions and fed ad libitum. Mature spermatozoa were extracted from the caudal epididymides and vas deferens of freshly killed 8-wk-old mice. The sperm-containing fluid was collected in 25 mM Tris and 150 mM KCl buffer (TKB) on ice for use in all experiments. All animal use was approved by the Animal Care and Use Committee of the University of Hawaii.
Sperm Cell DNA Fragmentation and Degradation Assays
Immediately after extraction, Triton X-100 (TX), a non-ionic detergent, was added to the spermatozoa suspended in TKB to a final concentration of 0.25% TX. Agarose plugs that were approximately 5 mm thick were made immediately by mixing 2% plugging agarose with the sperm sample to a final concentration of 1% agarose. This mixture was poured into molds and allowed to solidify. For the sperm chromatin fragmentation (SCF) assay, the individual plugs were then incubated in microfuge tubes containing TKB with various concentrations of cations and inhibitors, as described in Results. For the sperm DNA degradation (SDD) assay, the plugs were first extracted with 2 mM dithiothreitol (DTT) in TKB, or with 2 M NaCl, 2 mM DTT, and 25 mM Tris for 1 h. The NaCl/DTT incubation extracts the protamines while leaving the nuclear matrix and its associated proteins and DNA. The plugs were subsequently placed into microfuge tubes containing TKB with various concentrations of cations and inhibitors incubated at 37°C for various times. To stop the reaction, plugs were placed in digestion buffer (10 mM Tris, 5 mM EDTA, pH 7.8, 100 mM NaCl, 0.5% SDS, and 20 mM DTT) at 53°C for at least 30 min before placement in a 1% agarose gel for field inversion gel electrophoresis (FIGE).
Topoisomerase II Inhibition and Reversal of the Cleavable Complex
Agarose plugs containing spermatozoa that were treated to induce SCF or SDD as described above were incubated in 2 M NaCl or 10 mM EDTA for 15 min at 37°C to test for topoisomerase cleavage reversibility. The topoisomerase II-specific metabolic poison etoposide phosphate (EMD Biosciences) was suspended in H20 and used in a final concentration of 50 µg/ml in subsequent reactions with 5 mM ATP (Sigma). The topoisomerase II-specific metabolic inhibitor ICRF-193 (BioMol) was suspended in dimethyl sulfoxide (DMSO) and used at a final concentration of 1 µg/ml supplemented with 5 mM ATP. In experiments using topoisomerase II drugs, Tris was omitted from the NaCl/DTT extraction step.
Isolation of Spermatozoa from Epididymal Fluid
Mature spermatozoa were extracted as described above in TKB, washed, and centrifuged at least four times for 5 min each at 700 x g to separate the sperm cells from the surrounding fluid. The supernatant from the first separation was saved as spermatozoa-free epididymal fluid. The next wash was supplemented with TX to 0.25%, and the pellet was centrifuged and washed one more time before being aliquoted and pelleted for resuspension. The pellet of spermatozoa devoid of epididymal fluid was then resuspended in either epididymal fluid or TKB alone, and TX was added to a final concentration of 0.25%. Agarose plugs were made, and the SCF assay was performed as described above.
Field Inversion Gel Electrophoresis
Agarose plugs containing reaction products were applied to 1% agarose gels and run on a FIGE-Mapper FIGE system (BioRad) according to the manufacturer's protocol, and they were programmed to best resolve the DNA below 140 kb.
Immunoblot and Immunofluorescence Antibodies
Polyclonal antibodies against TOP2B (H-286; Santa Cruz Biotechnology) were used 1:250 for immunoblots. Secondary antibodies for immunoblots were anti-rabbit, alkaline phosphatase labeled (1:1000; Sigma) and were developed on polyvinylidene fluoride (PVDF) membranes using NBT/BCIP (Pierce). Spermatozoa were kept at 4°C in the presence of soybean trypsin inhibitor throughout the experiments.
RESULTS
Sperm DNA Fragmentation is Reversible, Suggesting Mediation by Topoisomerase II
We previously reported that mature spermatozoa contain the innate ability to digest their own DNA into loop-sized fragments of 50 kb after washing with TX and incubation with MgCl2 [5, 9]. Washing spermatozoa taken from the vas deferens and epididimydes of recently killed mature mice with 0.25% TX and incubating them with MnCl2 and CaCl2 for various times also induced the cell to fragment its DNA to 50 kb, but with much less time than our previous reports (Fig. 2, lanes 1–5). This appeared by FIGE to be complete by 4 h—overnight incubation did not elicit any new fragmentation (Fig. 2, lanes 4 and 5). This activity was readily reversible by incubation with 2 M NaCl for 15 min (Fig. 2, lanes 6–10) or 10 mM EDTA for 30 min (Fig. 2, lanes 11–15). Interestingly, this was true even in cells that had been incubated in MnCl2 and CaCl2 overnight. This salt or EDTA reversal of fragmented DNA is indicative of topoisomerase II activity [23, 24]. We term this degradation of sperm DNA to loop-sized fragments, which is reversible by EDTA, sperm chromatin fragmentation (SCF).
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TOP2B is Detected in Spermatozoa by Immunoblot
The reversibility of SCF suggested the involvement of an active TOP2B in mature spermatozoa. We tested for the presence of TOP2B by immunoblot detection with a polyclonal antibody to human TOP2B that is known to cross-react with mouse TOP2B. Spermatozoa, with and without the epididymal fluid and in the presence of soybean trypsin inhibitor to protect against proteolytic degradation, contain a high-molecular weight protein that reacts with the TOP2B-specific antibody (Fig. 3). This detected protein is the expected molecular weight of TOP2B and also is detected in spleen homogenate. The fluid in which the sperm cells were collected does not contain TOP2B. All of these data together show that TOP2B is present in mature spermatozoa and absent from epididymal and vas deferens fluid.
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Extraction with Salt and DTT Revealed a Second, Irreversible Nuclease Activity
TOP2B is associated with the nuclear matrix in somatic cells and retains its activity after the 2 M NaCl extraction used to prepare matrices [25]. We tested whether the reversible SCF activity also was present in sperm nuclear matrices. Treatment of spermatozoa with 2 M NaCl and 2 mM DTT extracts the protamines and many other proteins not bound to the sperm nuclear matrix but maintains the organization of DNA into loop domains by the nuclear matrix (Fig. 1) [8]. When the TX-washed sperm cells were first extracted with NaCl/DTT and then incubated with MnCl2 and CaCl2, DNA fragmentation to 50 kb was still present (Fig. 4, lane 2). However, in NaCl/DTT-extracted cells, an additional activity became evident; this was the complete digestion of DNA (Fig. 4, lanes 1–5). We term this complete digestion sperm DNA degradation (SDD). After 15 min in MnCl2 and CaCl2, some of the DNA fragmentation could be reversed by 2 M NaCl (Fig. 4, lane 7). Longer incubation times in MnCl2 and CaCl2, however, rendered the fragmentation of DNA irreversible (Fig. 4, lanes 9, 10, 14, and 15). This suggested the involvement of a second enzyme, a nuclease that digested the DNA in an irreversible manner.
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After 4 h, the digestion of sperm DNA was complete, suggesting that the nuclease was capable of digesting all of the DNA. Surprisingly, treatment with EDTA, which reversed TOP2B-induced DNA fragmentation, consistently appeared to accelerate SDD (Fig. 4, compare lanes 4 and 14; see also Fig. 5). In this and subsequent experiments we found that NaCl/DTT extraction of protamines prior to addition of MnCl2 and CaCl2 also increased the rate of the DNA fragmentation reaction (compare lanes 1–5 in Figs. 2 and 4). In sperm cells treated with NaCl/DTT the 50-kb product was visible by 15 min, whereas cells not treated with NaCl/DTT required more than 1 h to exhibit SCF. The SDD reaction also was incomplete or absent in cells not extracted with NaCl/DTT, whereas it was complete in salt-extracted cells.
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Nuclease Activity Requires DTT
DTT in combination with salt is required to remove protamines. However, this reducing reagent has a wide range of effects, including activating caspases that have been shown to activate apoptotic nucleases [26, 27]. We therefore tested whether DTT alone, without salt, could activate the nuclease activity. Spermatozoa incubated in TX and then DTT prior to addition of MnCl2 and CaCl2 cleaved their DNA to slightly smaller fragments than those without DTT, but this fragmentation was not reversible (Fig. 5, lanes 1–5). These data suggested that the nuclease activity requires DTT, but protamines may prevent the nuclease from digesting the DNA to completion.
Divalent Cation Requirements of SCF and SDD
To elucidate the requirements for divalent cations for each of these activities, experiments were carried out in NaCl-extracted cells and DTT-extracted cells so that both the SCF and SDD activities would be present. We previously showed that incubation in MgCl2 could induce SCF, but this required incubation times of 20 h [5]. However, in NaCl/DTT-extracted cells we found that MnCl2 was more efficient at inducing this activity—more so than MgCl2, CaCl2, or ZnCl2, alone (Fig. 6A). MgCl2 in combination with CaCl2 activated SCF but not SDD (Fig. 6B, lanes 1–4). MnCl2 alone activated SCF but not SDD (Fig. 6A, lane 2), but MnCl2 in combination with CaCl2 reproducibly activated both SCF and SDD (Fig. 6B, lanes 5–7). Because MnCl2 was the most active, all experiments were performed using this divalent cation. We next titrated the MnCl2 and CaCl2 together and independently (Fig. 7). Reducing both cations to 1.25 mM retarded the complete digestion of the sperm DNA, but the fragmentation to 50 kb was still evident (Fig. 7A, lanes 1–7). This suggested that the SCF activity was still present, whereas the SDD was not induced. With both cations absent from the reaction, neither activity was detected (lanes 8–10).
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Next we evaluated the effects of keeping the concentration of MnCl2 stable at 10 mM while varying CaCl2. There was almost no change in SCF and only a slight retardation of SDD in CaCl2 concentrations down to 1.25 mM (Fig. 7B, lanes 1–7). In reactions lacking CaCl2, only SCF was activated—DNA was not degraded beyond the fragmented 50-kb point (Fig. 7B, lanes 8–10). Decreasing the MnCl2 concentration while the CaCl2 concentration remained constant decreased the rate at which DNA was fragmented to 50 kb by 15 min (Fig. 7C, lanes 2 and 5). Despite the lowered MnCl2 concentration, by 1 h some of the fragmentation was evident, yet there was clearly a slowing of the DNA degradation activity (Fig. 7C, lanes 3 and 6). By 4 h, the DNA degradation below 50 kb was reduced proportionally to the concentration of MnCl2 (Fig. 7C, lanes 4 and 7). Again, CaCl2 alone was unable to support either the SCF or the SDD reaction (Fig. 7C, lanes 8–10).
Inhibition of SDF and SDD with TOP2B Inhibitors
The data in Figures 2 and 3 suggested that TOP2B mediated the first part of the reaction, degrading the DNA to 50-kb fragments. We tested this with two specific inhibitors of topoisomerase II. The first antitopoisomerase II drug is the catalytic inhibitor ICRF-193. This drug is specific for the enzyme's ATPase domains, preventing any DNA cleavage by TOP2B [28]. Adding ICRF-193 to spermatozoa inhibited SCF, the fragmentation of DNA to 50 kb through 4 hours (Fig. 8A). This provides further evidence that TOP2B mediates SCF.
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The second inhibitor, etoposide, is a so-called "topoisomerase II poison," because it stabilizes the covalent DNA-TOP2B reaction intermediate (the "cleavable complex") [29]. In the cleavable complex, the TOP2B monomers remain bound to each other, and each one is covalently attached to one broken end of the cleaved, double-stranded DNA. When the reaction is stopped with SDS-containing digestion buffer, the Topo is denatured. This splits the Topo into separated monomers that are still covalently linked to the broken DNA ends [13, 14]. We found that when sperm nuclear matrices were treated with MnCl2 and CaCl2 in the presence of etoposide, the reaction that normally proceeded rapidly to complete digestion (Fig. 8B, lanes 1–4) was now slowed significantly, with a drastic increase in the 50-kb DNA population (Fig. 8B, lanes 5–7). These data suggested that when the TOP2B was arrested at the cleavable complex stage the nuclease could not digest the DNA. Etoposide inhibits SDD but not SCF.
SCF and SDD are both Regulated by a Factor in the Epididymal Fluid
The data suggest that TOP2B mediates SCF and is involved in SDD, but they also support the existence of another nuclease that can completely digest the DNA during SDD. NaCl-extracted sperm and DTT-extracted sperm also were boiled before the SCF/SDD reaction, and all DNA remained unaffected (data not shown). These data show that the enzymes causing each reaction, SCF or SDD, indeed are contained within the mature sperm cells or the epididymal fluid and are not a product of a reaction contaminant. To determine whether the enzymes that regulated SCF and or SDD were present in the sperm cell or in the surrounding epididymal fluid, we thoroughly washed spermatozoa with TKB buffer by centrifugation and then tested for SCF and SDD by incubating the sperm with MnCl2 and CaCl2 after extracting the protamines with salt and DTT. We found that both activities were absent in sperm cells washed more than four times (Fig. 9A, lane 4).
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This absence suggested that a factor that regulates both SCF and SDD was located in the epididymal fluid and could be completely removed from the spermatozoa. We tested whether we could reconstitute SCF with the epididymal fluid. Resuspending washed spermatozoa in epididymal fluid generated the 50-kb DNA cleavage pattern typical of unwashed sperm cells (Fig. 9A, lane 3), whereas resuspending the washed spermatozoa in buffer alone had no effect (Fig. 9A, lane 4). These data suggest that the TOP2B-dependent cleavage of sperm DNA to 50 kb requires a factor in the epididymal fluid.
Epididymal Fluid Contains a Mn2+-Dependent and Ca2+-Dependant Nuclease
We have shown that spermatozoa require both Mn2+ and Ca2+ to completely degrade their DNA (Figs. 6B and 7B). Because the activation of degradation of sperm DNA required the epididymal fluid, we tested the possibility that there was an extracelluar nuclease in the epididymal fluid with the same ion requirements as SDD. Spermatozoa were cleared from the fluid by centrifugation. Different metals then were added to the epididymal fluid in the presence of 1 µg plasmid DNA to assay for nuclease activity. The addition of 10 mM Mn2+ and Ca2+ was required for complete nucleolytic digestion of the DNA; Mg2+, or Mn2+ or Ca2+ separately, were ineffective at stimulating DNA digestion (Fig. 9B). We showed that ICRF specifically inhibits TOP2B (Fig. 8A) and that TOP2B is not present in epididymal fluid (Fig. 3). Adding ICRF-193 to the Mn2+ and Ca2+ nuclease assay did not inhibit nucleolytic digestion of plasmid DNA (Fig. 9B). These data show that the epididymal fluid contains an Mn2+-dependent and Ca2+-dependant nuclease.
DISCUSSION
Our data suggest that mature mouse spermatozoa contain an active TOP2B that regulates DNA degradation in association with an extracellular nuclease. We provide evidence for two distinct nucleolytic activities that are functionally related. In the first, SCF, TOP2B mediates DNA fragmentation to 50 kb, and this fragmentation is reversible. Even though SCF was mediated by the sperm nuclear TOP2B, it required the addition of a factor(s) from the epididymal fluid. The second activity, SDD, was the digestion of the DNA after SCF by the nuclease. The nuclease activity was inhibited by the topoisomerase II poison etoposide, suggesting that SCF and SDD are mechanistically associated.
Our data support the existence in spermatozoa of an active TOP2B that mediates SCF. The reversibility of double-stranded DNA breaks with salt or EDTA is a unique characteristic of topoisomerase II [23, 24] (Fig. 2). Furthermore, a catalytic inhibitor of TOP2B, ICFR-193, inhibited fragmentation (Fig. 7A). Also, immunodetection using antibodies specific for TOP2B identified an appropriately sized protein in spermatozoa (Fig. 8). Finally, the topoisomerase II poison, etoposide, had a surprising but reproducible inhibition on the DNA degradation by the nuclease. Etoposide treatment of untreated spermatozoa could not trap the cleavable complex (data not shown), suggesting that TOP2B was not actively cleaving and religating DNA, as it does in somatic cells. However, the inhibition of SDD by etoposide supports a role for TOP2B in the degradation of DNA by the nuclease (see below).
Our data also point to the existence in SDD of a nuclease that can digest the sperm DNA to completion. It is important to note that the nuclease activity required DTT for activation (Fig. 5). It is possible that this activity is related to the reduction of the protamine disulfides, but we think this is unlikely. We have previously shown that exogenous DNAse I can digest the toroid linker regions in the absence of protamine disulfide reduction by DTT [5]. This demonstrated that there are areas within mammalian sperm chromatin that are susceptible to nuclease digestion even when the protamines are fully disulfide linked. If the endogenous Mn/Ca-dependent nuclease required DTT only to remove protamines, it should have digested these linker regions irreversibly in the absence of DTT, but this did not happen (Fig. 2). We are currently investigating the molecular basis for the requirement of DTT in activating the nuclease activity.
Our interpretations of the reversible SCF and irreversible SDD shown in Figures 2, 4, and 5 are diagrammed in Fig. 10. During SCF, a factor from the epididymal fluid activates the sperm TOP2B to cleave all of the DNA to 50-kb fragments in a reversible manner (Fig. 10A). We hypothesize that this occurs on the sperm nuclear matrix at the bases of the DNA loop domains at the matrix attachment regions (MARs). We previously demonstrated that MARs are located in the DNAse-sensitive toroid linker regions [5]. When DTT is included in this reaction, the epididymal fluid nuclease is activated and digests the toroid linker regions, but it does not digest the DNA bound to protamines in toroids (Fig. 10B). These data suggest that once the endogenous Mn/Ca nuclease is activated with DTT treatment, it is inhibited by protamine toroids, just as exogenous DNAse I is. When the protamines were extracted with salt and DTT prior to adding the cations, the nuclease digested all the sperm DNA (Fig. 10C).
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We are currently pursuing the identity of the epididymal fluid nuclease that is responsible for SDD. At least two laboratories have reported the existence of DNASE1L3, a calcium-magnesium-dependent nuclease, in bull semen [30, 31]. This nuclease is normally associated with the chromatin of somatic cells, and bull semen is the only reported extracellular existence of this enzyme. DNASE1L3 requires Mg2+ and Ca2+ for activation and can induce DNA fragmentation in somatic cells in a Ca2+-dependent and Mg2+-dependant fashion [32]. Even though SDD is much more active with Mn2+ than with Mg2+, it is possible that the SDD nuclease is DNASE1L3 or a closely related enzyme. We attempted to use the only commercially available monoclonal antibody to DNASE1L3, but this did not react with mouse epididymal fluid. However, this antibody recognizes the likely nuclear localization peptide on the enzyme—a sequence that may be cleaved off if DNASE1L3 is, in fact, extracellular [33]. Regardless of whether or not the SDD nuclease is DNASE1L3, it does appear to be an extracellular nuclease that interacts with intracellular TOP2B. Another nuclease is detectable in the epididymus of Dnase1-deficient mice [34], but it is unclear whether this is extracellular.
We are currently investigating several potential models for the mechanisms of SCF and SDD. Our data suggest at least two components of this mechanism. First, an extracellular factor in the epididymal fluid, perhaps the nuclease itself, activates TOP2B to cleave all of the DNA during SCF. Second, the data suggest that sperm TOP2B and the extracellular nuclease interact to digest the DNA in a controlled manner. This is supported by our data showing that the topoisomerase II-specific poison, etoposide, inhibits the nuclease digestion of DNA in SDD, suggesting a close interaction between these two enzymes. Furthermore, in somatic cells TOP2B has been shown to bind directly to another apoptotic nuclease, DFFB [11], and, more recently, TOP2B was shown to interact directly with DNA damage and repair enzymes [35]. This inhibition of SDD by etoposide also implies that before the nuclease can digest the DNA during SDD, the TOP2B must religate the cleaved DNA. The fact that EDTA, which promotes TOP2B religation, also increases the speed of DNA digestion during SDD further supports this component of the model. It is unclear why this religation step occurs, but it might serve as a checkpoint mechanism for the destruction of the sperm DNA. If this is true, the treatment with EDTA may actually lead to a progression of the mechanism toward SDD rather than the complete reversal depicted in Figure 10A. While the model is still under investigation, these data indicate it is a highly regulated mechanism.
How might the topoisomerase and nuclease function in vivo? One possibility is that the topoisomerase and nuclease might be part of an apoptotic mechanism in the sperm cell. It is interesting to speculate that spermatozoa may have evolved a specialized apoptotic mechanism that physically isolated the nuclease from the sperm nucleus. Current work in our laboratory has shown that the nuclease activity we demonstrated (Fig. 9B) can be pelleted by a high-speed centrifugation of sperm-depleted epididymal fluid. This suggests the possibility that the epididymal nuclease is present in extracellular vesicles or complexes. While this may protect the sperm DNA from the nuclease, fortuitously it also affords us a unique system in which to study the TOP2B-nuclease interaction that has been so difficult to study in somatic cells, in which the two enzymes both are present in the nucleus. If so, it is a highly regulated mechanism in which the nuclease is sequestered outside the cell and enters only under specific circumstances. This would protect the sperm cell from easily digesting its DNA while at the same time allowing for a mechanism to degrade damaged spermatozoa that appear during the transit through the epididymus. A similar mechanism for the degradation of spermatozoa after androgen withdrawal recently was proposed by Jones [36]. Moreover, spermatozoa are coated in a "death cocoon" by the fibrinogenlike protein FGL2 in the epididymis [37], providing further evidence for highly regulated sperm apoptosis in the epididymis. It is possible that SDD is normally activated in such death cocoons, digesting the toroid linker regions in an irreversible manner, whereas the protamines maintain the highly condensed state of the sperm chromatin (Fig. 10B).
Another possibility that is not mutually exclusive is that TOP2B plays a role in normal embryogenesis and that the major function of the nuclease may be to serve a protective role by digesting exogenous DNA [38]. Since the chromatin of the mature spermatozoa is in a highly condensed and fairly inactive state, the topoisomerase likely is positioned on the nuclear matrix from condensation during spermatogenesis and is readily available for DNA decondensation in early embryogenesis. This sperm TOP2B also may be important for DNA replication of the paternal pronucleus before syngamy. We showed that the extracellular nuclease does not degrade endogenous DNA without the TOP2B acting first, thus possibly providing a checkpoint for complete sperm degradation. We also demonstrated that the nuclease does not digest sperm DNA unless activated by DTT. The Mn/Ca-dependent nuclease in the epididymal fluid, however, does digest plasmid DNA in the absence of DTT, suggesting another mechanism to protect the sperm DNA from uncontrolled nucleolytic digestion. In this model, the nuclease may serve two functions: digesting extracellular DNA to protect the sperm, and—only if SDD is activated—digesting the sperm DNA in an irreversible manner. Finally, at least one group has suggested that extracellular nucleases in the semen may help spermatozoa penetrate neutrophil aggregation during fertilization [39].
One important limitation of this work is that the conditions used to activate both TOP2B and the nuclease were not physiologic, containing relatively high concentrations of MnCl2 and CaCl2, as well as permeabilization with TX. Much lower concentrations of divalent cations could activate SCF, but this still required TX (Fig. 7A, lanes 6 and 7). These conditions were used here because they activated SCF and SDD quickly enough for us to analyze the activity in vitro. We recently have completed a more physiologic study in which spermatozoa were incubated with MnCl2 and CaCl2 in HCZB [40], a buffer used in mouse intracytoplasmic sperm injection (ICSI) studies, in the absence of TX, and we showed that sperm DNA is degraded in the oocyte (unpublished data).
It is possible that the sperm DNA digestion by an extracellular nuclease we describe here is an artifact that results from TX permeablization, but we view this as unlikely for two reasons. First, the nuclease digestion was inhibited by etoposide, suggesting that TOP2B may regulate the nuclease. We previously showed that exogenous DNAse I can digest sperm DNA at the toroid linker regions, so there is nothing to inhibit unregulated nucleases from damaging sperm DNA. Second, current ICSI studies in our laboratory suggest that this nuclease activity is even more highly regulated than is demonstrated in this work. When spermatozoa that have been treated with MnCl2 and CaCl2 in the physiologic buffer HCZB [40] without TX are injected into oocytes, the paternal pronucleus develops normally with all of its DNA intact. However, at about 6–7 h after injection, the paternal DNA degrades, while the maternal DNA, which is not degraded, is being replicated (unpublished data). These preliminary results suggest the possibility that the nuclease enters the oocyte with the sperm cell but is not activated to degrade the paternal DNA until the time when the embryo begins its first DNA replication. Whether or not the TOP2B-nuclease interaction that we have described is part of an apoptotic mechanism similar to that in somatic cells, the separation of these two enzymes has provided a useful model for studying this interaction. Finally, even though the exact physiologic role of the nuclease has yet to be determined, our data have demonstrated that mammalian spermatozoa contain an active TOP2B.
ACKNOWLEDGMENTS
We thank Dr. Stefan Moisyadi for valuable discussions of enzyme chemistry.
FOOTNOTES
2 Correspondence: FAX: 808 956 7316; wward{at}hawaii.edu 
1 Supported by NIH grant HD28501 to W.S.W. and by the Victoria S. and Bradley L. Geist Foundation.
Received: 29 June 2006.
First decision: 26 July 2006.
Accepted: 10 August 2006.
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 R. P. Martins and S. A. Krawetz Decondensing the protamine domain for transcription PNAS, May 15, 2007; 104(20): 8340 - 8345. [Abstract] [Full Text] [PDF]
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 D. T. Carrell, B. R. Emery, and S. Hammoud Altered protamine expression and diminished spermatogenesis: what is the link? Hum. Reprod. Update, May 1, 2007; 13(3): 313 - 327. [Abstract] [Full Text] [PDF]
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Y. Yamauchi, J. A. Shaman, and W. S. Ward Topoisomerase II-Mediated Breaks in Spermatozoa Cause the Specific Degradation of Paternal DNA in Fertilized Oocytes Biol Reprod, April 1, 2007; 76(4): 666 - 672. [Abstract] [Full Text] [PDF]
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