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Topoisomerase II-Mediated Breaks in Spermatozoa Cause the Specific Degradation of Paternal DNA in Fertilized Oocytes¹

Institute for Biogenesis Research, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii 96822

ABSTRACT

We have demonstrated that mouse spermatozoa can cleave their DNA into 50-kb fragments when treated with Triton X-100, MnCl₂, and CaCl₂. This cleavage, which is termed sperm chromatin fragmentation (SCF), is mediated by topoisomerase IIB (TOP2B) following stimulation by a factor in the epididymal fluid, most likely a nuclease, and can be at least partially religated by EDTA. When the protamines are removed, this DNA breakage is followed by digestion of the DNA by a nuclease(s). We tested whether the oocyte could repair TOP2B-induced sperm DNA breaks and whether partial religation by EDTA would allow spermatozoa to fertilize the oocytes normally. Oocytes injected with untreated spermatozoa developed normally. However, oocytes injected with spermatozoa treated with MnCl₂ and CaCl₂ to induce SCF, with or without subsequent EDTA treatment, failed to develop. In both of these treatment groups, the maternal pronuclei developed normally and replicated their DNA. However the paternal pronuclei did not replicate their DNA and this DNA began to disappear 6 h postinjection, which corresponded approximately to the time at which maternal DNA replication was initiated. These data suggest that when TOP2B is induced to cleave sperm DNA before fertilization, the paternal DNA is subsequently degraded by a highly regulated mechanism that does not affect the maternal chromatin. Furthermore, partial religation by EDTA of TOP2B-induced breaks prevents neither the inhibition of DNA synthesis nor DNA degradation.

apoptosis, early development, fertilization, gamete biology, sperm

INTRODUCTION

Mammalian sperm DNA is tightly compacted by protamines, which condense the chromatin into toroids, each of which contains approximately 50 kb of DNA [1, 2]. This chromatin is also organized into loop domains of about the same size that are attached at their bases to the sperm nuclear matrix [3, 4]. We have proposed a Donut-Loop model for sperm chromatin structure in which each protamine toroid represents a single DNA loop domain [5, 6]. Between each toroid is a DNase I-accessible span of DNA, called the toroid linker region, which is also the point at which the DNA loop domains are attached to the nuclear matrix. DNase I treatment of spermatozoa releases the DNA loop domains without digesting them further [5]. This model supports the idea that sperm chromatin may contain so-called ‘active domains' or foci in which the DNA is not as condensed as in the protamine toroids [7].

We have recently provided evidence to support the idea that these attachment sites of the highly condensed sperm DNA may be more active than the rest of the chromatin. Mouse spermatozoa that are incubated with Triton X-100, MnCl₂, and CaCl₂ degrade their entire DNA to 50-kb fragments. This fragmentation is mediated by an extracellular factor, most likely a nuclease, which activates the sperm nuclear TOP2B, and is reversible by EDTA [8]. We term this reversible DNA fragmentation sperm chromatin fragmentation (SCF). When the protamines are first removed by salt and dithiothreitol (DTT), the fragmented DNA is subsequently completely degraded. This process is very similar to DNA degradation during somatic cell apoptosis [9–11]. In somatic cells, TOP2A or TOP2B, depending on the cell type, is associated with the nuclear matrix. During apoptosis, the DNA is first cleaved at this point of attachment [9, 12]. We have suggested that in SCF, TOP2B is associated with the DNA loop domain attachment sites in the DNase I-sensitive toroid linkers, in a similar manner [8].

SCF is an interesting model for studying the DNA degradation that occurs in apoptotic-like processes, since it provides a natural biological assay for the integrity of sperm DNA. This is because spermatozoa can be injected into oocytes to test for DNA repair based on successful embryonic development. In the present study, we used SCF to study the role of TOP2B in this process. In particular, we tested whether the oocyte has the ability to repair the TOP2B-induced sperm DNA breaks in SCF and whether partial EDTA religation of the TOP2B breaks allows the oocyte to repair sufficiently the sperm DNA so as to allow the embryo to develop normally after fertilization.

MATERIALS AND METHODS

Animals

B6D2F1 (C57BL/6J x DBA/2) mice were obtained at 6 weeks of age from National Cancer Institute (Raleigh, NC). All mice used in this study were fed ad libitum with a standard diet and maintained in a temperature (22°C) and light-controlled (14L:10D) room, 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 (DHEF publication no. [NIH] 80–23, revised 1985). 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.

Collection of Oocytes

Mature 8- to 12-wk-old females 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 [13]. The cumulus-oocyte complexes were released from the oviducts into 0.1% bovine testicular hyaluronidase (300 USP units/mg) in HCZB medium, to disperse the cumulus cells. The cumulus-free oocytes were washed with HCZB medium and used immediately for intracytoplasmic sperm injection (ICSI).

Collection of Spermatozoa and the Sperm Chromatin Fragmentation Assay

Mature spermatozoa were extracted from the caudal epididymides and vas deferens of freshly killed B6D2F1 mice (approximately 8 weeks old) and collected in HCZB for normal ICSI or in modified HCZB (mHCZB; HCZB that lacked Mg²⁺ and EDTA) for the SCF assay. Immediately after collection, spermatozoa for the assay were separated into three groups: Control, SCF, and SCF-Religated. Control spermatozoa were incubated at room temperature for 1–2.5 h. In both the SCF and SCF-Reversal groups, 10 mM MnCl₂ and 10 mM CaCl₂ were added to the sample and incubated at room temperature for 1.5 h for complete chromatin fragmentation. At 1.5 h, the SCF-religated group was supplemented with 100 mM EDTA to religate topoisomerase-induced strand breaks. To stop the reaction and resolve the DNA fragmentation, samples were imbedded in agarose plugs, incubated in digestion buffer, and separated by Field Inversion Gel Electrophoresis (FIGE) as previously described [8]. For microinjection of the treated sperm into mouse oocytes, 2 µl of the sperm sample was transferred into 200 µl of HCZB instead of plugging in agarose.

Intracytoplasmic Sperm Injection

ICSI was carried out as described recently by Szczygiel and Yanagimachi [14]. Briefly, a small drop of treated sperm suspension was mixed thoroughly with an equal volume of HCZB that contained 12% (w/v) polyvinyl pyrollidone (PVP, 360 kDa) immediately before ICSI. ICSI was performed using Eppendorf Micromanipulators (Micromanipulator TransferMan; Eppendorf, 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.

Enucleation of Metaphase II Oocytes

Enucleation of metaphase II oocytes was performed as described previously [15]. Briefly, groups of oocytes (usually 10–15) were transferred into a droplet of Hepes–CZB that contained 5 µg /ml cytochalasin B, which had previously been placed in the operation chamber on the microscope stage. After the oocyte was held by the holding pipette, its zona pellucida was ‘drilled’ by applying several Piezo pulses to the tip of the enucleation pipette (10-µm inner diameter). The metaphase II chromosome-spindle complex was drawn into the pipette with a minimal volume of oocyte cytoplasm. After enucleation of all the oocytes in one group (approximately 10 min), they were transferred into cytochalasin B-free CZB and incubated for up to 2 h at 37°C, then returned to the microscope stage immediately before sperm injection. Following sperm injection, oocytes were placed immediately in Ca²⁺-free CZB that contained 10 mM Sr²⁺ and incubated for 4 h. Following activation, the embryos were washed and transferred to Sr²⁺-free CZB medium and incubation was continued at 37°C under 5% CO₂ in air.

Embryo Culture

After ICSI, the oocytes were cultured in 50-µl droplets of CZB (HCZB without Hepes buffer) overlaid with mineral oil for 5 h at 37°C in 5% CO₂ in air. The oocytes with two distinct pronuclei and the second polar body were considered to be normally fertilized. Fertilized eggs were cultured in 50 µl of CZB under the same conditions. Cultured eggs were evaluated for developmental progress at 24 h and 96 h after ICSI. The survival rate of oocytes after sperm injection ranged from 69.2% to 78.6% in all four groups.

Assessment of DNA Replication

DNA replication analysis was performed according to the procedure described earlier [16] with modifications [17]. Normally fertilized oocytes (with two polar bodies and two pronuclei) were incubated in CZB that contained 10 µM 5-bromo-2-deoxyuridine (BrdU) for 30 min at several time-points after ICSI. Following incubation in BrdU, the oocytes were fixed in a solution made by adding 0.5M NaOH and 2.5% paraformaldehyde to PBS (pH 7.3) for 15 min at room temperature. Fixed oocytes were washed in PBS that contained 10% fetal bovine serum (FBS) and 0.2% Triton X-100 and blocked in the same solution for 30 min at 37°C. The oocytes were then washed in PBS that contained 2% FBS and 0.1% Triton X-100. The oocytes were then incubated in drops of Alexa Fluor 488-conjugated anti-BrdU antibody (Invitrogen), diluted 1:19 in PBS that contained 2% FBS and 0.1% Triton X-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 that contained propidium iodide (Vector Laboratories, Burlingame, CA) and examined using a fluorescence microscope that was fitted with the appropriate filters.

Statistical Analysis

Comparisons of fertilization, embryo development, and DNA replication rates following ICSI were carried out using the chi-square test.

RESULTS

Sperm DNA Fragmentation Occurs in HCZB Without the Addition of Triton X-100

To prepare SCF-treated spermatozoa for injection into oocytes, we first tested whether SCF could be activated in HCZB, which is the physiological buffer used for mouse ICSI [13]. We prepared a modified form of HCZB (mHCZB) that did not contain MgCl₂ or EDTA, since both affect SCF. Spermatozoa were incubated with mHCZB supplemented with MnCl₂ and CaCl₂ under various conditions, then plugged in agarose and electrophoresed by FIGE. We found that in mHCZB media, SCF could be induced in spermatozoa without the inclusion of Triton X-100 (Fig. 1A). Therefore, in subsequent experiments, we omitted Triton X-100 from the incubation buffer. Spermatozoa incubated for 1–2.5 h in mHCZB do not exhibit any DNA fragmentation, as assayed by FIGE (Fig. 1B, lane 1). The incubation of sperm cells in mHCZB supplemented with 10 mM Mn²⁺ and 10 mM Ca²⁺ induced SCF to degrade the entire DNA to loop-sized fragments that ranged in size between 50 kb and 20 kb by 1.5 h (Fig. 1B, lane 2). We have previously suggested that this degradation is the result of the sperm DNA being cleaved at the bases of the DNA loop domains [8]. Since eukaryotic DNA loop domain sizes are heterogeneous, ranging from 100 kb to 5 kb [18–21], degrading the DNA at the loop bases would result in a heterogeneous smear, such as that seen in Figure 1. Further incubation of Mn²⁺- and Ca²⁺-treated spermatozoa with 100 mM EDTA for 30 min caused the DNA fragments to religate partially (Fig. 1B, lane 3).

View larger version (86K): [in this window] [in a new window] [Download PPT slide]   FIG. 1. SCF occurs in mHCZB in the absence of Triton X-100. A) Mouse spermatozoa were incubated in mHCZB with (lanes 1–4) or without (lanes 5–8) 0.5% Triton X-100, and then incubated with MnCl2 and CaCl2 to induce SCF for various time periods. Samples were then embedded in agarose plugs, incubated in digestion buffer, and subjected to separation by FIGE. B) Mouse spermatozoa were incubated for 1–2.5 h in mHCZB (Control, lane 1) or in mHCZB supplemented with 10 mM MnCl2 and 10 mM CaCl2 (SCF, lane 2), to induce SCF. After incubation in MnCl2 and CaCl2, EDTA was added to 100 mM to an aliquot of spermatozoa, to religate the TOP2B-induced double strand breaks (SCF-Religated, lane 3). Samples were then embedded in agarose plugs, incubated in digestion buffer, and subjected to separation by FIGE.

Oocytes Injected with Spermatozoa Possessing Fragmented or Subsequently Partially Religated DNA Are Unable to Develop Past the 2-Cell Stage

We next tested whether the oocyte could repair TOP2B-mediated DNA breaks and whether reversing TOP2B-mediated sperm chromatin fragmentation with EDTA before injection could repair the sperm chromatin sufficiently to allow for normal embryo development. We first prepared four different treatment groups for injection: (1) spermatozoa collected in traditional HCZB (normal control); (2) spermatozoa incubated for 1–2.5 h in modified HCZB without divalent cation supplementation (treatment control); (3) SCF spermatozoa incubated in 10 mM Mn²⁺ and 10 mM Ca²⁺ to induce SCF; and (4) SCF-religated spermatozoa incubated in 10 mM Mn²⁺ and 10 mM Ca²⁺, followed by 30 min in 100 mM EDTA to religate the TOP2B-induced breaks. We followed the development of the embryos through the blastocyst stage in each of the four groups. In each ICSI experiment, the spermatozoa that were injected were tested for normal DNA, SCF, and SCF reversal by gel electrophoresis (Fig. 1B).

Both control groups developed normally, which demonstrates that additional manipulation of spermatozoa is required to induce SCF, and that using mHCZB instead of HCZB has no detrimental effects on embryo development (Table 1). When spermatozoa were induced to fragment their DNA, fewer than 50% of the embryos developed to the two-cell stage, and only 10.8% developed to the blastocyst stage. This suggests that the embryo is not efficient at repairing TOP2B-mediated DNA breaks. When the sperm DNA fragmentation was partially religated with EDTA before injection, the frequency of development was slightly worse (6.7%). This suggests that partial religation of TOP2B-induced DNA breaks by EDTA does not repair the DNA sufficiently to allow the spermatozoa to participate in normal embryonic development.

SCF Causes Inhibition of Paternal DNA Replication and Eventual Degradation of the Paternal DNA

We also attempted to determine why the embryos injected with SCF-activated spermatozoa or with EDTA-religated spermatozoa failed to develop. Oocytes were injected with spermatozoa from the treatment groups listed above and then incubated with BrdU to observe DNA synthesis in the pronuclei. The embryos were counterstained with propidium iodide to detect DNA. In control samples viewed at 7 h postinjection, the maternal and paternal pronuclei appeared to be normal, with DNA replication and homogeneous DNA staining detected in both (Fig. 2, A–C). In oocytes injected with SCF-induced spermatozoa, the maternal pronuclei appeared to have normal DNA replication and homogeneous DNA staining. However, no DNA replication was evident in the male pronucleus. In fact, the propidium iodide staining was much weaker, suggesting that the paternal DNA had been degraded (Fig. 2, G–I). Interestingly, the paternal pronucleus was clearly visible by phase microscopy even though the paternal DNA had been degraded (Fig. 2G). In 49 oocytes examined by this method, 69% had normal maternal pronuclear DNA replication, while only 10% of the paternal pronuclei exhibited visible DNA replication, and 64% of the paternal pronuclei showed evidence of DNA degradation after 7 h (Table 2). Embryos produced by injection with EDTA-religated spermatozoa showed very similar results (Fig. 2, J–L and Table 2).

View larger version (78K): [in this window] [in a new window] [Download PPT slide]   FIG. 2. DNA synthesis and degradation in TOP2B-cleaved and EDTA-religated paternal pronuclei. Oocytes were injected with control or treated spermatozoa and incubated for 9 h (or 5 h, D–F) at 37°C. The samples were then incubated with BrdU for 30 min, subsequently fixed and fluorescently labeled with anti-BrdU antibodies (B, E, H, K), and the DNA was counterstained with propidium iodide (C, F, I, L). A–C) Control; normal untreated spermatozoa. D–F) SCF; spermatozoa incubated in 10 mM MnCl2 and CaCl2 for 1.5 h to induce SCF, and then tested for DNA synthesis at 5 h postinjection. G–I) SCF tested 9 h postinjection. J–L) SCF-Religated; MnCl2- and CaCl2-treated spermatozoa incubated with 100 mM EDTA for 30 min, to religate the TOP2B-induced cleavage. Phase contrast images are also shown (A, D, G, J). Bar = 20 µm.

As a control for these experiments, we injected enucleated oocytes with treated spermatozoa, to determine if the absence of the maternal pronucleus had any affect on DNA synthesis inhibition or paternal DNA degradation. As shown in Figure 3, we obtained the same results with enucleated oocytes as with intact oocytes, thereby confirming that the pronuclei with inhibited DNA synthesis and DNA degradation (Fig. 2) are the paternal pronuclei. Control, parthenogenetically activated oocytes replicated their DNA normally (Fig. 3B), as did enucleated oocytes that were injected with control spermatozoa (Fig. 3E). Spermatozoa treated with MnCl₂ and CaCl₂ with or without subsequent EDTA treatment did not replicate their DNA (Fig. 3, G–L), and they began to degrade their DNA by 7 h postinjection (Table 3).

View larger version (70K): [in this window] [in a new window] [Download PPT slide]   FIG. 3. DNA synthesis and degradation in TOP2B-cleaved and EDTA-religated paternal pronuclei injected into enucleated oocytes. Oocytes were enucleated and then injected with control or treated spermatozoa and incubated for 7 h at 37°C. They were then incubated with BrdU for 30 min, fixed and fluorescently labeled with anti-BrdU antibodies (B, E, H, K), and the DNA was counterstained with propidium iodide (C, F, I, L). A–C) Control oocytes that were not enucleated and subsequently parthenogenetically activated. D–F) Control; spermatozoa incubated with mHCZB for 1.5 h without MnCl2 and CaCl2. G–I) SCF; spermatozoa incubated in 10 mM MnCl2 and 10 mM CaCl2 for 1.5 h to induce SCF, and then tested for DNA synthesis at 7 h postinjection. J–L) SCF-Religated; MnCl2- and CaCl2-treated spermatozoa incubated with 100 mM EDTA for 30 min to religate the TOP2B-induced cleavage. Phase contrast images are also shown (A, D, G, J). Bar = 20 µm.

We next examined more closely the paternal DNA replication and degradation. We observed that embryos from oocytes injected with SCF-induced spermatozoa developed normal pronuclei and retained their entire DNA for up to 5 h after injection, as compared to normal controls (Fig. 2, C and F). After 6 h, the percentage of embryos that displayed evidence of DNA replication in both the control and SCF-induced groups started to increase (Fig. 4A). Control groups that showed male and female pronuclei were positive for DNA replication, while in the majority of SCF-induced embryos, only the maternal DNA was replicated. In the SCF-induced group, the percentage of embryos with normal paternal pronuclear DNA staining began to decrease (Fig. 2, G–I, and 3B). By 7 h postfertilization, most of the embryos showed some evidence of paternal DNA degradation, whereas the maternal DNA remained intact (Figs. 2I and 4B). These data suggest that paternal DNA degradation postfertilization is not activated by decondensation but by an event that occurs much later.

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   FIG. 4. Timing of DNA replication and degradation in SCF-initiated embryos. A) DNA replication in the male and female pronuclei of control and SCF-initiated embryos was examined over time after injection of BrdU. For control embryos, only those that have both male and female pronuclei labeled with BrdU are shown (black diamonds), as most of the embryos fall into this category. For SCF-induced embryos, those with both male and female pronuclei labeled (grey squares) and those with only the female pronucleus labeled (grey triangles) are shown. B) The percentage of embryos that contain a normal amount of DNA, as determined by PI staining of SCF-induced embryos, was determined over time. In all cases, the female pronuclei had normal PI staining. For each time-point in both graphs, more than 20 zygotes were analyzed, except for the 5-h time-point, for which there are nine zygotes in A and 16 zygotes in B.

DISCUSSION

Our data suggest that spermatozoa that have been induced to fragment their DNA into loop-sized fragments by SCF are no longer capable of supporting embryonic development when injected into oocytes. Since fragmentation is mediated by TOP2B, which can religate the DNA breaks it creates [22, 23], it is mechanistically possible that the oocyte can repair all the paternal DNA breaks. Therefore, to control for this possibility, we tested whether these embryos could undergo development. Although we did not test directly whether all or even some of the paternal DNA breaks were repaired by the oocyte, the embryos did not develop past the two-cell stage, the paternal DNA was not replicated, and the paternal DNA was degraded 7–9 h after injection. Therefore, the zygotes that resulted from injection with SCF-induced spermatozoa could not repair the damage sufficiently for the first round of DNA synthesis or further development to proceed.

When EDTA was used to induce TOP2B to religate the DNA before injection, the embryos also did not develop, even though the DNA appeared to be at least partially religated in the FIGE analysis. As with the oocytes that were injected with SCF-induced spermatozoa, the embryos injected with SCF-religated spermatozoa degraded and did not replicate their paternal DNA after 6 h. This suggests that even when the TOP2B-induced DNA breaks are partially religated, the oocyte cannot repair the damage sufficiently to allow for normal development or DNA synthesis to occur. However, it is also possible that EDTA treatment did not religate all the DNA breaks induced by SCF; even if it did, the DNA strands that were religated may not have been religated correctly.

The paternal DNA in embryos injected with SCF or SCF-religated spermatozoa was not degraded until more than 7 h after injection. In fact, the paternal pronuclei appeared to develop normally, and were even larger than the maternal pronuclei, as seen in normal mouse embryos [24]. This suggests that at least in the oocyte, paternal DNA degradation by the nuclease is not induced by protamine replacement by histones, which occurs at the end of chromatin recondensation, before the fully formed pronucleus is apparent [25, 26]. Our previous in vitro experiments have suggested that when SCF is induced by Triton X-100 in the presence of Mn²⁺ and Ca²⁺, the DNA is not degraded completely unless DTT is included, which suggests that the protamines inhibit the nuclease. However, the 6-h lag in paternal DNA degradation in vivo suggests that some other factor regulates paternal DNA degradation in the oocyte. The identity of this factor remains unknown, although we may speculate that it is related to the initiation of DNA synthesis, since the timing of paternal DNA degradation corresponds roughly to that of maternal DNA synthesis.

Paternal DNA degradation and the inhibition of DNA synthesis in zygotes that result from injecting oocytes with SCF or SCF-religated spermatozoa appear to be very specific for the paternal pronucleus. Most of the maternal pronuclei replicated their DNA in these embryos and none of the maternal pronuclei exhibited evidence of DNA degradation. This suggests that the nuclease that digests the paternal DNA and the factor(s) that prevents paternal DNA synthesis are contained within the pronuclei. It is interesting that the paternal pronucleus remained clearly visible by phase microscopy, even after the DNA was degraded, indicating that the pronuclear structure does not depend on the continued presence of DNA. We have suggested that TOP2B is located on the sperm nuclear matrix at the sites of DNA attachment [8], as has been shown in somatic cells by several laboratories [27–29], and that during SCF, the nuclease associates with TOP2B at these attachment sites. Such compartmentalization of the nuclease with the nuclear matrix would restrict the digestion of the DNA to the paternal pronucleus, as shown in the present study. It is also possible that an oocyte-specific nuclease recognizes the TOP2B-induced DNA breaks in the paternal pronucleus, and specifically degrades the paternal DNA. In either case, the sperm TOP2B-induced DNA breaks that initiate the degradation probably limit complete digestion to the paternal chromatin.

In our first report on SCF, we suggested a physiological role for this sperm DNA degradation process whereby the nuclease is sequestered outside the sperm cell and is translocated to the sperm nucleus upon activation of SCF [8]. However, in order to study SCF, we used Triton X-100 for maximal stimulation. In the present report, we found that in a more physiologically relevant buffer (HCZB), Triton X-100 caused the reaction to proceed too quickly, and we omitted it from subsequent studies. That a detergent is not required for SCF supports our suggestion of a physiological role for SCF. Furthermore, we found that while paternal DNA replication was inhibited in almost all of the zygotes from SCF or SCF-religated sperm ICSI, only about half of the zygotes showed clear evidence of paternal DNA degradation (Table 2). This suggests the possibility that one physiological action of SCF is to induce TOP2B cleavage of sperm DNA to inhibit DNA synthesis. This type of mechanism is consistent with one hypothesis regarding the function of sperm DNA degradation, which is that degradation prevents spermatozoa that have somehow acquired DNA damage during spermiogenesis or fertilization from successfully fertilizing an oocyte [30].

Finally, we have not yet identified the nuclease that is involved in sperm DNA degradation and that appears to be associated with SCF. The present study supports the possibility that when SCF is activated, a nuclease enters the sperm cell, which can then be carried into the oocyte during ICSI. However, it is equally possible that an oocyte-specific nuclease recognizes the TOP2B breaks induced by SCF. These data suggest that sperm chromatin can be modified under near-physiological conditions in such a way that it prevents early development after fertilization. Our data also support the model of sperm chromatin structure in which "active domains" are maintained even in the highly condensed sperm chromatin. Furthermore, while it is possible for SCF-mediated sperm DNA degradation to occur outside of the oocyte, as seen in our in vitro studies, some of the processes can clearly be regulated in vivo by factors in the newly fertilized zygote.

ACKNOWLEDGMENTS

The authors acknowledge Dr. Monika A. Ward for suggesting the enucleation experiments as a control and for technical advice regarding these experiments.

FOOTNOTES

³These authors contributed equally to this work.

¹Supported by NIH 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 Road, Honolulu, HI 96822. FAX: 808 956 7316; e-mail: wward{at}hawaii.edu

Received: 2 September 2006.

First decision: 18 October 2006.

Accepted: 15 December 2006.

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