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Institute for Biogenesis Research, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii 96822
ABSTRACT
We recently demonstrated that mouse spermatozoa contain a mechanism to degrade their DNA into loop-sized fragments of about 50 kb, mediated by topoisomerase IIB, termed sperm chromatin fragmentation (SCF). SCF is often followed by a more complete digestion of the DNA with a sperm nuclease. When SCF-induced spermatozoa are injected into oocytes, the paternal pronuclei degrade their DNA after the initiation of DNA synthesis, but the maternal pronuclei are unaffected and replicate normally. Here, we tested whether the nuclease activity changes in spermatozoa of different maturation stages, and whether there is a functional relationship between the initiation of DNA synthesis and paternal DNA degradation induced by SCF in the zygote. We found that spermatozoa from the vas deferens have a much higher level of SCF activity than those from the cauda epididymis, suggesting that spermatozoa may acquire this activity in the vas deferens. Furthermore, paternal pronuclei formed in zygotes from injecting oocytes with SCF-induced vas deferens spermatozoa degraded their DNA, but this degradation could be inhibited by the DNA synthesis inhibitor, aphidicolin. Upon release from a 4 h aphidicolin-induced arrest, DNA synthesis was initiated in maternal pronuclei, while the paternal pronuclei degraded their DNA. Longer aphidicolin arrest resulted in the paternal pronuclei replicating their DNA, suggesting that delaying the initiation of DNA synthesis allowed the paternal pronuclei to overcome the SCF-induced DNA degradation pathway. These results suggest that the paternal DNA degradation, in oocytes fertilized with SCF-induced spermatozoa, is coupled to the initiation of DNA synthesis in newly fertilized zygotes.
early development, epididymis, sperm maturation, vas deferens
Many studies have shown that spermatozoa from several different mammalian species contain DNA double-stranded breaks when the animals are exposed to environmental or chemical stresses [1–5]. In humans, the incidence of DNA breaks in sperm chromatin has been linked to poor fertility, but the source of these breaks remains unclear [6–9]. While single-stranded DNA breaks are more common and may correlate better to male infertility than double-stranded breaks, the latter can cause more permanent, less easily reparable, damage. One suggested possibility is that DNA breaks in fully mature spermatozoa are the result of apoptotic events that occurred during spermatogenesis [10, 11].
We have recently suggested another possible mechanism for the induction of DNA breaks in sperm chromatin. Mouse spermatozoa that are incubated with MnCl2 and CaCl2 actively degrade their own DNA. This is a two-step process in which topoisomerase IIB (TOP2B) first cleaves the DNA to loop-sized fragments of roughly 50 kb, which are then degraded by a sperm nuclease [12]. Sperm chromatin is condensed into tightly compacted toroids by protamines that are resistant to nucleases and other DNA-damaging agents [13]. However, this endogenous TOP2B/nuclease degradation of the sperm DNA is initiated at the nuclease-sensitive sites of DNA attachment to the nuclear matrix [14, 15]. Spermatozoa treated with MnCl2 and CaCl2 undergo this reaction very quickly in vitro, but when they are injected into oocytes, the paternal DNA seems to remain intact through the pronuclear stage [16]—the paternal pronucleus forms normally and the DNA appears to remain intact by propidium iodide staining for the first 6 h after injection. Shortly afterwards, the male pronucleus begins to degrade its DNA, while the female pronucleus is unaffected and initiates normal DNA replication. This suggests the possibility that the paternal DNA degradation that results from TOP2B DNA breaks is regulated by the initiation of DNA synthesis.
This type of DNA degradation in spermatozoa has many similarities to that of DNA degradation in somatic cells. During apoptosis, eukaryotic cells initiate a TOP2-mediated fragmentation of the chromatin into loop-sized pieces on the nuclear matrix [17–19]. This is followed by the more complete digestion of the DNA by various nucleases that appear to be closely associated with TOP2 [20, 21]. There are also precedents for the relationship between the cell's DNA damage recognition and repair mechanisms with DNA synthesis. One example is that DNA damage can induce the arrest of DNA synthesis through p53-dependent pathways [22, 23]. Here, the role of the DNA synthesis arrest is to allow the cell time to repair the damage before DNA synthesis resumes and, if it is not repaired, to direct the cell toward apoptosis [24, 25]. Recently, a more direct relationship between DNA damage and synthesis has been shown in the DNA repair protein, RecA, which requires the initiation of DNA synthesis before it responds to the damage [26].
The role of such mechanisms in the spermatozoon and in the paternal pronucleus is not yet clear. We have suggested that they might exist as a checkpoint to prevent DNA-damaged spermatozoa from introducing mutations to the developing embryo [27]. Regardless of the reason, it is clear that mammalian spermatozoa contain a mechanism for DNA degradation, and this mechanism initiates a regulated response in the fertilized oocyte. The timing of the paternal DNA degradation correlates well with the initiation of DNA synthesis in the zygote [13]. In this work, we tested whether the degradation of the paternal DNA in zygotes that were injected with DNA containing double-stranded breaks required the initiation of DNA synthesis.
B6D2F1 (C57BL/6J x DBA/2) mice were obtained from the National Cancer Institute (Raleigh, NC). Mice were kept in accordance with the guidelines of the Laboratory Animal Services at the University of Hawaii and those prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Resources National Research Council. The protocol for animal handling and the treatment procedures were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Hawaii.
Analysis of Sperm DNA Degradation by Field Inversion Gel Electrophoresis
Plasma from the caudal epididymides and vas deferens of 
8-wk-old mice was extracted separately and suspended in mHCZB (modified Hepes-CZB, CZB media buffered with Hepes and without magnesium, calcium or EDTA) [16], to a final concentration of about 108 sperm/ml. The suspension was mixed with agarose to a final concentration of 1% agarose and poured into molds making 5-mm-thick plugs. The plugs were incubated at 37°C in TKB (25 mM Tris, pH 7.4, 150 mM KCl) supplemented with 10 mM MnCl2 and 10 mM CaCl2 for 0 time, 15 min, 1 h, or 4 h to initiate sperm chromatin fragmentation or DNA degradation. For each time point, one plug was incubated in digestion buffer at 55°C to stop the reaction, and one plug was incubated for 30 min with 30 mM EDTA to religate topoisomerase-induced strand breaks before stopping the reaction [12]. After the reaction, the plugs were incubated in digestion buffer (10 mM Tris, 5 mM EDTA, pH 7.8, 100 mM NaCl, 0.5% SDS, and 20 mM dithiothreitol) at 53°C for at least 30 min before placement in a 1% agarose gel for field inversion gel electrophoresis (FIGE).
Preparation of Spermatozoa for Intracytoplasmic Sperm Injection
Spermatozoa were collected in mHCZB, as described above, and divided into three groups: control, SCF, and SCF-religated. Control spermatozoa were incubated at room temperature for 1–2.5 h. In the SCF and SCF-religated groups, spermatozoa were incubated at room temperature for 1 h in the presence of 10 mM MnCl2 and 10 mM CaCl2. After 1 h, the SCF-religated group was supplemented with 100 mM EDTA. Treated spermatozoa were injected immediately after the reaction's time was reached.
Mature females, 8- to 12-wk-old, were induced to superovulate with i.p. injections of 5 IU eCG and 5 IU hCG given 48 h apart. Oviducts were removed 14–15 h after the injection of hCG and placed in HCZB [28]. The cumulus-oocyte complexes were released from the oviducts into 0.1% bovine testicular hyaluronidase (300 USP units/mg) in HCZB medium to disperse cumulus cells. The cumulus-free oocytes were washed with HCZB medium and used immediately for intracytoplasmic sperm injection (ICSI).
Spermatozoa prepared for ICSI were mixed with 12% (w/v) polyvinyl pyrollidone (PVP; 360 kDa); single sperm heads were injected into each oocyte. ICSI was carried out as described recently by Szczygiel and Yanagimachi [29]. A small drop of treated sperm suspension was mixed thoroughly with an equal volume of HCZB containing 12% (w/v) PVP (Mr, 360 kDa) immediately before ICSI. ICSI was performed using Eppendorf micromanipulators (Micromanipulator TransferMan; Eppendorf AG, Hamburg, Germany) with a Piezo-electric actuator (PMM Controller, model PMAS-CT150; Prime Tech, Tsukuba, Japan). A single spermatozoon was drawn, tail first, into the injection pipette and moved back and forth until the head-midpiece junction (the neck) was at the opening of the injection pipette. The head was separated from the midpiece by applying one or more piezo pulses. After discarding the midpiece and tail, the head was redrawn into the pipette and injected immediately into an oocyte.
Embryo Culture and Aphidicolin Treatment
After ICSI, oocytes were cultured in CZB (e.g., CZB that is not buffered with Hepes or modified [29]) for 5 h at 37°C in 5% CO2 in air. For aphidicolin treatment, oocytes were transferred to fresh media containing 3 µg/ml aphidicolin (Sigma) [30] 3 h after ICSI for an additional 4 or 15 h. Aphidicolin was prepared as a 1 mg/ml stock solution in dimethyl sulfoxide (DMSO) and stored at –20°C until used. For use, a 3-µl aliquot was diluted into 997 µl CZB. Oocytes were released from aphidicolin by transferring to fresh media without the drug. Oocytes with two distinct pronuclei and a second polar body were considered to be normally fertilized. Cultured eggs were evaluated for developmental progress at 24 and 96 h after ICSI.
DNA replication analysis was performed according to the procedure described previously [31] with modifications [32]. Normally fertilized oocytes (with two polar bodies and two pronuclei) were incubated in CZB with 10 µM 5-bromo-2-deoxyuridine (BrdU) for 30 min at several time points after ICSI. BrdU was prepared as a 1-mM stock solution in DMSO, and 10 µl was added to 990 µl of media for BrdU incubation. Following incubation in BrdU, the oocytes were fixed with 2.5% paraformaldehyde in PBS (with 0.5 M NaOH, pH 7.3) for 15 min at room temperature. Fixed oocytes were washed in PBS containing 10% fetal bovine serum (FBS) and 0.2% Triton X-100 (TX-100) and blocked in the same solution for 30 min at 37°C. The oocytes were then washed in PBS containing 2% FBS and 0.1% TX-100 (PBS: 2% FBS/0.1% TX-100). Oocytes were then incubated in drops of anti-BrdU antibody conjugated with Alexa Fluor 488 (Invitrogen) and diluted 1:19 in PBS (2% FBS/0.1% TX-100) for 1 h at 37°C. The oocytes were placed on poly-lysine (1 mg/ml)-coated microscope slides. The preparations were covered with VectaShield mounting media containing propidium iodide (Vector Laboratories, Burlingame, CA) and examined using a fluorescence microscope (Nikon Eclipse E600 at x200 magnification) fitted with the appropriate filters (000). Maternal and paternal pronuclei were differentiated by the fact that the paternal pronucleus in the mouse is larger [33]. We verified our ability to identify male oocytes by size in our last study by creating oocytes with only maternal pronuclei, by parthenogenetic activation, or only paternal pronuclei, by injecting SCF-induced spermatozoa into enucleated oocytes [16].
Spermatozoa from the Vas Deferens Have More SCF Activity
In our two previous studies on the TOP2B-mediated fragmentation of sperm DNA, we combined spermatozoa from the cauda epididimydes and from the vas deferens [12, 16]. However, we have found recently that, when treated with divalent cations, vas deferens spermatozoa have a much higher level of DNA degradation activity. We induced SCF independently in spermatozoa from epididymal and vas deferens plasma by incubation in mHCZB with MnCl2 and CaCl2. Epididymal sperm DNA was rapidly (within 15 min) fragmented to 50 kb (Fig. 1A, lanes 1–8). This fragmented DNA religated with EDTA incubation, forming high molecular weight DNA similar to that in control sperm cells. Interestingly, after the first 15 min, this fragmentation appears to autoreligate without EDTA treatment—the fragmentation to 50 kb that existed at 15 min is reduced at 1 h, and further reduced at 4 h. This suggests that, in epididymal spermatozoa, only the TOP2B-mediated cleavage is activated. In contrast, sperm DNA from vas deferens was rapidly degraded after the 15 min treatment with MnCl2 and CaCl2, and minimally reversed after subsequent EDTA incubation (Fig. 1A, lanes 9–14). The fragments were also much smaller than in the epididymal spermatozoa. This suggests that the nuclease is much more active in vas deferens spermatozoa than in epididymal spermatozoa.
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Oocytes Injected with Vas Deferens, but Not with Epididymal SCF-Induced Spermatozoa, Degrade Their Paternal DNA
Because of the unexpected differences between the ability of epididymal and vas deferens spermatozoa to degrade their DNA by SCF, we repeated our previous experiments in which we injected SCF-induced spermatozoa into oocytes [16]. The schemes for these and the following experiments are diagrammed in Figure 2. For all experiments, FIGE analyses were performed to ensure that DNA fragmentation by SCF had occurred (examples are shown in Fig. 1B). We first injected either control (untreated) or SCF-induced epididymal or vas deferens spermatozoa into oocytes. We then cultured the zygotes for 7 h, and added BrdU to the media for 30 min, before fixing and staining for BrdU incorporation (Fig. 2B). We used the 30-min BrdU incubation time in order to compare our results to our previous findings, in which we found that paternal pronuclei formed from injection of SCF-induced spermatozoa degraded, and did not replicate, their DNA [16]. We found that epididymally-derived SCF spermatozoa supported maternal and paternal pronuclear formation in mouse oocytes (Fig. 3, A–C). DNA replication was evident in 90% of oocytes with 2 pronuclei, which was similar to control injections, and 97% of paternal pronuclei showed no evidence of DNA degradation (Table 1); however, only 15% of these embryos developed to the blastocyst stage (Table 2). Oocytes that were injected with SCF-induced epididymal sperm, which were treated with EDTA to religate the DNA fragmentation, developed in a similar manner to those injected without EDTA-treated spermatozoa—they replicated their paternal DNA (Table 1) but did not develop (Table 2).
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We next injected SCF-induced sperm cells from the vas deferens. These supported maternal and paternal pronuclear formation in the oocyte (Fig. 3, D–F). However, at 7 h postinjection, there was no detectable paternal DNA replication in 78% of the oocytes where maternal replication was detected (Table 1). Furthermore, the paternal DNA had been degraded in 70% of the paternal pronuclei in oocytes, while the maternal DNA remained intact. Oocytes injected with EDTA-treated SCF spermatozoa from the vas deferens yielded similar results. Neither group developed to the blastocyst stage (Table 2). Zygotes formed from normal, control, vas deferens sperm injection replicated their paternal DNA normally (Table 2). These controls also developed to the blastocyst stage, although with a slightly lower percentage of embryos than control, epididymal sperm injected oocytes (Table 2).
Finally, because we incubated zygotes in BrdU for only 30 min, we may have missed a small amount of DNA replication that could have occurred in the paternal pronucleus at the initiation of DNA synthesis. DNA replication in mouse single-cell zygotes occurs over 7 h [34], and the 30-min BrdU incubation in the previous experiments occurred in the middle of the DNA synthesis stage [32]. However, it is possible that the paternal pronucleus had initiated DNA synthesis earlier and stopped before we added BrdU at 7 h postinjection, and this newly replicated DNA was not degraded. To control for this, and as a control for the following studies where we had to use longer BrdU incubation times, we repeated the experiments with vas deferens spermatozoa, but incubated the zygotes with BrdU for 4 h, beginning well before DNA synthesis was initiated (Fig. 2B). The results were identical to those shown in Figure 3 (D–F)—there was no detectable BrdU incorporation in the paternal pronucleus, and the female pronucleus was still labeled (Table 1).
These results suggest that the paternal DNA degradation that we had previously reported by performing ICSI with SCF-induced spermatozoa was largely due to the vas deferens spermatozoa in our samples. Because we were focusing on the relationship between DNA replication and DNA degradation, we used only vas deferens spermatozoa for the rest of this study.
Inhibiting DNA Replication Successfully Inhibits Male Pronuclear DNA Degradation
We recently demonstrated that the paternal pronuclear DNA degradation in zygotes that were formed by injecting SCF-induced spermatozoa into oocytes occurs at approximately the same time as the initiation of DNA replication [16]. We therefore tested whether this degradation was dependent upon DNA synthesis. Oocytes were injected with untreated or SCF-induced spermatozoa, cultured for 3 h, and then transferred to media containing aphidicolin for an additional 4 h (Fig. 2C). In the mouse, DNA synthesis of both pronuclei begins between 5 and 6 h after ICSI [32] (Fig. 2A), so, in this experiment, aphidicolin was present well before its initiation. After the incubation in aphidicolin, the oocytes were transferred to media containing BrdU for 30 min, then fixed and stained. In control spermatozoa-injected oocytes, DNA synthesis was inhibited in the maternal and paternal pronuclei. In oocytes injected with SCF-induced vas deferens spermatozoa, the maternal pronucleus did not replicate its DNA as it normally does, indicating that aphidicolin inhibited DNA replication (Fig. 3, G–I, and Table 3). The paternal pronucleus of these oocytes also did not replicate its DNA; however, the paternal DNA was not degraded by 7 h, as normally occurs in these oocytes. These data indicate that SCF-induced paternal DNA degradation in the zygote can be inhibited by inhibiting DNA synthesis.
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Release from Aphidicolin Inhibition Allows Some DNA Synthesis to Occur
We next examined whether releasing the inhibition of DNA replication would also allow the paternal pronuclear degradation to continue. To test this, we injected untreated and SCF-induced vas deferens spermatozoa into oocytes, cultured them for 3 h, and then transferred the zygotes to aphidicolin containing media for 4 h (Fig. 2D). The zygotes were then transferred to fresh media to release them from DNA synthesis inhibition, and incubated with BrdU for an additional 4 h. When control zygotes injected with untreated vas deferens spermatozoa were released from aphidicolin, both pronuclei replicated their DNA within 4 h (Fig. 3, J–L, and Table 3). These zygotes developed into blastocysts at a slightly lower rate than control nuclei without aphidicolin treatment and release (Table 4). This indicated that mouse embryos could survive a brief aphidicolin arrest and still develop normally. Zygotes from SCF-induced sperm injection replicated their maternal DNA normally and degraded their paternal DNA within the same time period when released from aphidicolin (Table 3). This supports our previous work indicating that the paternal and maternal nuclei are independently regulated with respect to DNA synthesis and degradation [16]. As expected, none of these embryos developed to the blastocyst stage (Fig. 3O and Table 4). Thus, release from DNA synthesis inhibition also released paternal DNA degradation inhibition.
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These zygotes, however, also exhibited a faint but detectable and reproducible intensity of BrdU labeling in the paternal pronucleus as compared with the female pronucleus (Fig. 3N and Table 3). This low level of BrdU labeling was not seen in SCF zygotes that were not treated with aphidicolin (Fig. 3E and Table 3). These data suggest that aphidicolin arrest overcame some of the inhibition of DNA synthesis elicited by SCF. We tested this by incubating SCF zygotes for a longer period of time in aphidicolin. Untreated and SCF-induced spermatozoa were injected into oocytes, which were cultured for 3 h and then arrested with aphidicolin for 15 h before release (Fig. 2D). In this case, neither the zygotes from control nor SCF-induced sperm injection developed past the two-cell stage (Table 4), indicating that mouse embryos cannot survive such a prolonged block in aphidicolin. However, maternal and paternal pronuclei of control and experimental zygotes initiated DNA replication, and had similar levels of BrdU incorporation in each pronucleus (Fig. 3, Q and T). The pronuclei appeared slightly distorted in control and experimental zygotes, but the initiation of DNA replication in both groups was clear. Moreover, none of the paternal pronuclei degraded their DNA (Fig. 3U and Table 3). This suggests that prolonged time in DNA synthesis arrest allows the paternal pronucleus to overcome the DNA degradation signal from SCF-induced spermatozoa. There is a gradual increase in the number of oocytes, with similar levels of BrdU incorporation into both pronuclei, with increasing time of incubation in aphidicolin (Fig. 4).
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These results demonstrate that the degradation of the paternal DNA in oocytes injected with SCF-induced spermatozoa occurs only after the initiation of DNA synthesis. This suggests that the signals in the embryo cytoplasm that normally signal both pronuclei to replicate also serve to signal the paternal pronucleus to degrade its DNA when SCF sperm are injected by ICSI. This degradation occurs after the protamines are removed and replaced by histones and the sperm decondenses into a pronucleus [13] (Fig. 3). This suggests that the regulation of the paternal DNA degradation is more than simply the release of the tightly compacted sperm chromatin by the embryo. Inhibition with aphidicolin prevented the degradation from occurring for up to 18 h after ICSI, demonstrating that paternal DNA degradation requires some components of DNA synthesis.
Spermatozoa from the vas deferens had a much greater ability to degrade its DNA than those from the epididymis (Fig. 1). This suggests that the sperm cell acquires the ability to degrade its DNA during its transit through the end of the cauda epididymis or in the vas deferens, itself. In the epididymis, the sperm cell matures and develops the potential to fertilize. This maturation has been widely studied (for reviews, see [35, 36]). In addition to the absorption of membrane proteins, the sperm cell chromatin is also altered by oxidation of protamine sulfhydryls to form disulfide bonds that further stabilize the compacted DNA [37]. Recently, it has been shown that alterations in protamine deposition due to the lack of transition proteins results in increased paternal chromatin aberrations when spermatozoa from the cauda epididymis are used for ICSI as compared with those from the caput epididymis [38]. However, it was not known whether the source of these aberrations occurred in the spermatozoon or in the oocyte after ICSI. Although very little has been written about the possible maturation of sperm chromatin in the vas deferens, at least two reports have suggested that chromatin changes do occur at this stage [39, 40]. The work presented here suggests that at least one aspect of sperm maturation may occur in the vas deferens: the ability to degrade its DNA in the presence of the appropriate environmental signal.
We are currently evaluating the differences seen here between epididymal and vas deferens spermatozoa, and it appears to be the result of higher concentrations of the nuclease in the latter (data not shown). That even the epididymal spermatozoa contain a low level of nuclease is supported by the finding in this work that, while the paternal pronuclei of zygotes formed from SCF-induced epididymal spermatozoa replicated their DNA, these zygotes did not develop (Table 2). This suggests that even these spermatozoa had subtle, irreversible chromosomal breaks caused by the nuclease that prevented normal development. Furthermore, in these experiments, significantly fewer oocytes injected with untreated vas deferens spermatozoa developed to the blastocyst stage (60%) than those injected with epididymal spermatozoa (81%; Table 2). This lower rate of development in ICSI using normal vas deferens spermatozoa raises the possibility that, in the vas deferens control group, some SCF was activated even without the addition of MnCl2 and CaCl2. In fact, the males used in this study were never mated, and it may be that the increased susceptibility to the induction of SCF in vas deferens is the result of time in the duct. If so, this would suggest that SCF may be part of a normal sperm-clearing mechanism of "unused" spermatozoa. At least one other study has suggested that vas deferens spermatozoa are less able to fertilize than epididymal spermatozoa, and proposed that the vas deferens spermatozoa had aged [41]. Further experiments would have to be preformed to determine whether this trend is demonstrable, but it suggests that the epididymis may prove to be a slightly better source of spermatozoa for ICSI than the vas deferens.
These experiments also demonstrate that, when SCF-induced vas deferens spermatozoa are injected into oocytes, the paternal degradation that occurs 6 h after fertilization requires DNA synthesis. When DNA synthesis was inhibited by aphidicolin, the paternal DNA degradation was also inhibited (Fig. 3, G–I). Releasing the zygotes from 4 h of aphidicolin inhibition allowed controls to develop to the blastocyst stage, although at a lower rate than for untreated controls (42% [Table 4] vs. 60% [Table 2], respectively), demonstrating that mouse embryos can survive a short aphidicolin treatment. Zygotes resulting from ICSI with SCF-induced vas deferens spermatozoa initiated delayed DNA synthesis in the maternal pronuclei and delayed paternal DNA degradation (Fig. 3, M–O). Aphidicolin inhibits all three DNA polymerases associated with eukaryotic DNA replication—POLA1, POLD1, and POLE [42, 43]—and its presence in cells before the initiation of DNA synthesis blocks replication forks at their origins [44, 45]. The fact that aphidicolin inhibited paternal DNA degradation therefore suggests that this digestion is dependent on the start of DNA synthesis.
Release from 4 h of aphidicolin treatment resulted in low levels of BrdU incorporation in the paternal pronucleus that was not seen in other treatments. This suggests that some of the ability to replicate DNA was restored to the paternal pronucleus, but only at a low level. The DNA still appeared to be degraded in these same zygotes. After releasing the oocytes from 15 h of aphidicolin treatment, the paternal pronucleus appeared to have restored most of its ability to replicate its DNA (Fig. 4). The pronuclei of both control (Fig. 3, P–R) and SCF zygotes (Fig. 3, S–U) appeared slightly distorted, but the DNA replication machinery evidently survived the overnight inhibition. The amount of BrdU incorporation into the paternal DNA of SCF zygotes appeared to be slightly less than that of the maternal DNA in SCF zygotes (Fig. 3T), suggesting that less DNA synthesis occurred. Interestingly, however, after 15 h in aphidicolin, none of the paternal pronuclei appeared to degrade their DNA. It is unclear how aphidicolin acts to allow the SCF sperm DNA to replicate, but one possibility that we are currently investigating is that the DNA breaks are repaired. Aphidicolin inhibits the DNA replication polymerases, but does not inhibit the DNA polymerase for repair, POLDB [46, 47]. It is therefore possible that prolonged aphidicolin treatment allows the embryo to repair enough of the sperm DNA damage for some DNA replication to proceed.
The DNA double-stranded breaks that are formed by SCF in the sperm cell are mediated by TOP2B, a nuclear matrix-associated protein [12, 48]. Also, we have recently demonstrated that, in the mouse, paternal DNA replication in the fertilized oocyte requires the sperm nuclear matrix [49]. Furthermore, sperm DNA breaks that are not associated with the sperm nuclear matrix do not result in the inhibition of DNA synthesis [49]. These data suggest that the foci within the sperm chromatin at which the DNA is directly associated with the nuclear matrix are actively involved in the regulation of the paternal DNA after fertilization. In somatic cells, DNA replication occurs at a fixed site on the nuclear matrix, and each loop domain is one replicon [50, 51]. Our data suggest that sperm and paternal pronuclear DNA degradation also occurs at the nuclear matrix. Therefore, it is plausible to consider that each replicon may also be a unit of DNA degradation by SCF. If so, one explanation for the data in Table 3 and in Figure 3, in which we saw DNA replication and degradation in the same pronucleus, may be that each DNA loop domain is individually regulated by the TOP2B-induced DNA breaks (Fig. 5F). In this model, the aphidicolin arrest allowed some of the loops with DNA breaks to replicate, while others are degraded.
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While we know that SCF in spermatozoa depends on TOP2B and a sperm nuclease [12], we do not yet know whether TOP2B-mediated double-stranded breaks are enough to signal the paternal pronucleus to degrade its DNA. It is possible, for example, that DNA synthesis is inhibited only if the nuclease subsequently digests some of the matrix-attached DNA. This would explain the difference between ICSI with SCF-induced spermatozoa from the epididymis and the vas deferens—when treated with MnCl2 and CaCl2 to induce SCF, epididymal spermatozoa appear to have mainly TOP2B breaks, while vas deferens spermatozoa exhibit some aspects of additional nuclease digestion (Fig. 1A). Alternatively, the TOP2B-mediated breaks may be enough to initiate paternal DNA degradation, and the differences between the two sperm populations may be quantitative. Future work in our laboratory will focus on developing more quantitative methods to analyze the levels of DNA degradation and replication in the zygotic pronuclei.
While the exact mechanism still needs to be elucidated, these data demonstrate that the temporal correlation between DNA synthesis and paternal DNA degradation in our previous work [16] is the result of a direct dependence of the degradation on DNA replication.
FOOTNOTES
3These authors contributed equally to this work. 
1Supported by National Institutes of Health grant HD28501 to W.S.W. and by the Victoria S. and Bradley L. Geist Foundation.
Correspondence: 2W. Steven Ward, Institute for Biogenesis Research, John A. Burns School of Medicine, University of Hawaii, 1960 East-West Rd., Honolulu, HI 96822. FAX: 808 956 7316; e-mail: wward{at}hawaii.edu
Received: 13 March 2007.
First decision: 17 April 2007.
Accepted: 8 May 2007.
REFERENCES
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