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From the Institute for Biogenesis Research, Department of Anatomy and Reproductive Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii.
| Correspondence to: Dr W. Steven Ward, Institute for Biogenesis Research, John A. Burns School of Medicine, University of Hawaii, 1960 East-West Road, Honolulu, HI 96822 (e-mail: wward{at}hawaii.edu). |
| Received for publication July 27, 2004; accepted for publication October 4, 2004. |
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Abstract |
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Key words: Sperm, nuclear matrix
The highly compact, inert state of sperm chromatin seems to conflict with these reports of its susceptibility to endogenous nucleases, but a recent model of sperm chromatin structure might provide a resolution. This model is based on earlier work by several laboratories. Balhorn and colleagues have demonstrated that the basic unit of protamine binding to DNA is the formation of a toroid that contains roughly 50 kb of DNA (Hud et al, 1993, 1994). We and others have shown that sperm DNA is organized into loop domains, also about 50 kb, that are attached at their bases to a proteinaceous nuclear matrix (Ward et al, 1989; Kalandadze et al, 1990; Kramer and Krawetz, 1996). On the basis of the similarity in size and of structural constraints, we proposed a model in which each loop domain was a single protamine toroid (Ward, 1993), which we term the donut loop model. We have recently provided supporting experimental evidence for this model from our work in hamster spermatozoa (Sotolongo et al, 2003). We showed that the DNA that links 2 consecutive protamine toroids to each other corresponds to matrix attachment regions (MARs) and is much more sensitive to exogenous DNAse I than the protamine-bound DNA. Thus, according to this model, most of the sperm DNA is highly condensed by protamines, but there are less compacted regions interspersed throughout the chromosome. These less compacted regions suggested by the donut loop model might be the sites of sperm chromatin digestions by endogenous nucleases.
Our recent report also suggested the existence of an endogenous sperm nuclease in hamsters that cleaved the DNA into loop-sized fragments of about 50 kb (Sotolongo et al, 2003). This nucleolitic activity resembles that of several nucleases in somatic cells that are activated during apoptosis and cleave the DNA into similar sizes (Li et al, 1999; Yakovlev et al, 2000; Boulares et al, 2002a,b; Solovyan et al, 2002). Our initial experiments, however, required overnight incubation of demembranated spermatozoa before the DNA cleavage was detectable. In this report, we show that the hamster sperm nuclease is much more active in the presence of both Ca2+ and Mg2+, cleaving sperm DNA within 30 minutes. Similar nucleases are also present in mouse and human spermatozoa.
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Materials and Methods |
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Preparation of Hamster Sperm Nuclei— Hamster sperm nuclei were prepared as previously described (Ward and Coffey, 1989). Briefly, hamster spermatozoa were sonicated to remove the tails then centrifuged through a step gradient consisting of the sonicate as the top layer; 2 M sucrose and 50 mM Tris, pH 7.4, as the second; and 0.40 mg/mL CsCl with 0.25% Triton X-100 as the third. The gradient was centrifuged at 100 000 x g for 45 minutes, and the pelleted nuclei were resuspended in 50 mM Tris, pH 7.4.
Preparation of Mouse Spermatozoa— Mouse spermatozoa were isolated by dissecting the epididymides and vas deferens from freshly euthanized males. The spermatozoa from the vas deferens were gently squeezed out by holding one end with forceps. The spermatozoa from the epididymides were then isolated by slicing the tissue and squeezing gently. All the spermatozoa were pooled in ice-cold 50 mM Tris, pH 7.4, and then counted with a hemacytometer. In a typical experiment, the spermatozoa from 10 mice were suspended in 4 mL Tris, with a concentration of about 3 x 107 spermatozoa/mL. This suspension was then made up to 0.25% TX100 and aliquotted, as described in the previous section. CaCl2, MgCl2, or both were added, as indicated, and the suspensions were incubated for various times at 37°C.
Preparation of Human Spermatozoa— Spermatozoa were obtained from semen of men presenting for routine infertility evaluation after an abstinence period ranging from 2 to 7 days. A standard semen analysis was performed within 30 minutes of sample collection, and only those normal according to the World Health Organization criteria (WHO, 1999) were included in the study. All specimens had counts of less than 1 million leukocytes/mL. Total sperm from ejaculates were prepared by 2x centrifugation and resuspension in HEPES-buffered human tubal fluid (HTF; Quinn et al, 1982) supplemented with 10% synthetic serum substitute (Irvine Scientific, Irvine, Calif). This commercial buffer was prepared from reagents free of nuclease activity (Irvine Scientific, personal communication). Alternatively, a sub-population highly enriched for normal motile sperm was prepared by centrifugation of semen over a 2-mL discontinuous gradient of PureCeption (40%:80%; Cooper Surgical, Trumbill, Conn) for 15 minutes at 1900 rpm in a clinical centrifuge, followed by resuspension of the pellet in 1 mL of HTF. This resulted in a population with more than 90% motility, more than 90% normal morphology, and no detectable round cells. Leukocytes do not penetrate the interface between the 40% and 80% layers in these preparations (Gandini et al, 1999). With either preparation, spermatozoa were used either fresh or frozen in liquid nitrogen without cryoprotectant and stored at -20°C. TX100 and MgCl2 were added for different time periods before pulsed-field gel electrophoresis (PFGE) analysis. CaCl2 was not added because this is a component of HTF. Osmolarity of all solutions was measured with an Osmette A osmometer from Precision Systems.
Pulsed-Field Gel Electrophoresis— After the spermatozoa were incubated and the nuclease digestions stopped with EDTA, as described before, the samples were mixed with equivalent amounts of 1% agarose and plugs were made. These PFGE plugs were incubated overnight at 53°C in lysis buffer adapted from Kozik et al (1998; 10 mM Tris-HCl, pH 8.0, 10 mM EDTA, 100 mM NaCl, 20 mM dithiotreitol [DTT], 2% sodium dodecyl sulfate [SDS], 20 µg/mL proteinase K). After incubation, the plugs were washed 3 times for 10 minutes, minimum, with 10 to 15 mL of TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) and 1 M glycine. Then they were washed with TE buffer alone 2 additional times. The plugs were stored at 4°C in TE buffer.
About 100 mL of a 1% PFGE agarose (BioRad Pulsed Field Certified Agarose, Hercules, Calif) in TBE (45 mM Tris-borate, 1 mM EDTA) was poured into the PFGE casting stand (14 x 13 cm) and mold after it had been tempered at around 55°C in a water bath for 10 minutes. The individual sample plugs were placed inside the wells of the gel and additional 1% PFGE agarose was used to seal the plugs in the well. The molecular mass marker was a low-molecular mass ladder (for pulsed field) made up of polymers of the 48.5-kb lambda phage genome (size range 48.5-291 kb in increments of 48.5 kb) and the HindIII-digested lambda phage genome (2-23 kb), making the entire range of 2-291 kb. The marker is provided in a syringe tube embedded in 1% agarose (New England Biolabs, Beverly, Mass). A small section of this agarose was squeezed out of the tube and cut off, and this small plug was embedded in the well.
The agarose gel with the standard casting platform frame and platform was placed into the Biorad PFGE Chef Mapper electrophoresis chamber and approximately 2.2 L of 0.5x autoclaved TBE was added. The temperature was allowed to equilibrate to 14°C, and the pump flow was set at 70 (about 1 L/min). The additional conditions for running the gel were as follows: linear ramping, 27 hours; 12 minutes total run time; 4 V/cm voltage; 120° angle, 50 kb to 1 mb range, initial switch time 6.75 seconds; final switch time, 33.69 seconds. After separation, the 1% agarose gel was then stained with ethidium bromide, and a Kodak EDAS 290 gel documentation picture was taken under ultraviolet light (Eastman Kodak Co, Rochester, NY).
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Results |
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The Endogenous Hamster Sperm Nuclease Requires Both Calcium and Magnesium for Full Activity— In our previous report, spermatozoa had to be incubated overnight after membrane disruption with TX100 before the activity of the endogenous sperm nuclease could be demonstrated (Sotolongo et al, 2003). Several reports in the literature describe the existence of nucleases in somatic cells that also cleaved DNA into large, loop-sized fragments of 20 to 100 kb that require both CaCl2 and MgCl2 for full activity (Lagarkova et al, 1995; Yakovlev et al, 2000; Boulares et al, 2002a, b). We therefore tested whether the hamster sperm nuclease also required both divalent cations for full activity. Hamster spermatozoa were washed with 0.25% TX100 and incubated for various amounts of time with a buffer containing either 10 mM Mg2+ alone, 10 mM CaCl2 alone, or both ions at 5 mM each. Control samples were incubated with 10 mM EDTA and did not contain Mg2+ or Ca2+. After incubation, the spermatozoa were embedded in agarose plugs and digested with SDS and proteinase K to remove all the proteins. Osmolarity measurements for the 3 solutions used—1) 50 mM Tris, pH 7.5, 0.25% TX100, 83 mOsm; 2) 50 mM Tris, 10 mM MgCl2, pH 7.5, 0.25% TX100, 99 mOsm; and 3) 50 mM Tris, 10 mM MgCl2, 10 mM CaCl2, pH 7.5, and 0.25% TX100, 109 mOsm—varied slightly, as one would predict by adding divalent cations. The plugs were then used for PFGE. As shown in Figure 1, spermatozoa that were incubated in the presence of Mg2+ alone required overnight incubation before the DNA was digested, as we have reported previously (Sotolongo et al, 2003). Spermatozoa incubated with Ca2+ alone appeared to digest their DNA faster but still required overnight incubation for most of the DNA to be digested. However, spermatozoa incubated with both cations digested their DNA to loop-sized fragments within 15 minutes. Treatment with both cations overnight often led to the complete digestion of sperm DNA, as shown in this example (Figure 1, last lane). These data are consistent with the presence of a sperm Ca/Mg-dependent nuclease.
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The Endogenous Hamster Sperm Nuclease is Present in the Sperm Nucleus—
At least 2 previous reports have described a Ca/Mg-dependent nuclease,
DNAS1L3 or DNAse 
, in bovine semen
(Hashida et al, 1982; Yakovlev et al, 1999). These
reports describe a purification method that does not preclude the possibility
that the nuclease was actually extracted from the bovine sperm nucleus. Other
reports describing this enzyme in somatic cells suggest that it is bound to
chromatin (Yakovlev et al,
2000; Boulares et al,
2002a,b).
In all of our previous work, the spermatozoa were obtained from epididymal
fluid that was diluted just before use. Because the epididymides provide some
of the seminal fluid, we could not preclude the possibility that the nuclease
was present in the hamster epididymal fluid. In addition, at least 1
Ca/Mg-dependent nuclease that also cleaves somatic cell DNA into loop-sized
fragments, Endo G, is present in mitochondria
(Zhang et al, 2003). We
therefore isolated hamster sperm nuclei to remove both the epididymal fluids
and the sperm tail mitochondria with sucrose step gradients, as described by
Ward and Coffey (1989), and
tested for the presence of the sperm nuclease we had previously described. As
shown in Figure 2, isolated
hamster sperm nuclei do contain the endogenous nuclease activity. Note that
although nuclease activity was apparent within 15 minutes in the sperm nuclei
(Figure 2), overnight
incubation did not result in complete digestion, as it did when whole
spermatozoa were incubated (Figure
1).
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Mouse Sperm Chromatin is Organized Into Donut Loops and Contains an Endogenous Nuclease— We next tested whether the chromatin organization of mouse spermatozoa was also consistent with our donut loop model, which provides the structural basis for nuclease digestion. The donut loop model, as described in the "Introduction," predicts that sperm chromatin treated with exogenous DNAse I will be digested into loop-sized fragments. This is because DNAse I digests the matrix attachment regions located between the toroids, but the rest of the DNA loop domain is protected by protamine binding. In these experiments, the spermatozoa were incubated with 10 mM MgCl2, but without CaCl2 so that only the exogenous DNAse I would be activated. As shown in Figure 3, mouse sperm nuclei treated with DNAse I are digested to loop-sized fragments. We next tested whether mouse spermatozoa contain the endogenous nuclease that cleaves sperm DNA into large fragments. Epididymal spermatozoa were washed with 0.25% TX100 and incubated in a buffer containing 5 mM each of both CaCl2 and MgCl2 for various times. As shown in Figure 4, the sperm DNA was digested into loop-sized fragments, just as in the hamster. Mouse spermatozoa, however, rarely digested their DNA completely after overnight digestion, as in the hamster. These data suggested that the mouse sperm chromatin is similar to that of the hamster in 2 important ways: the organization of DNA into donut loops and the existence of an endogenous Ca/Mg-dependent nuclease.
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Human Sperm Chromatin is Organized Into Donut Loops and Spermatozoa Contain an Endogenous Nuclease That is More Easily Activated Than the Rodent Nuclease— The donut loop model for sperm chromatin structure offered an explanation for the endogenous nuclease activity in both hamster (Sotolongo et al, 2003) and mouse (this study). We therefore attempted to demonstrate with exogenous DNAse I that human sperm chromatin had a similar arrangement. Figure 5 shows that treatment of human spermatozoa with various concentrations of DNAse I for only 15 minutes results in the 50-kb digestion pattern. However, longer incubation times with DNAse I caused more complete digestion that was not seen in the mouse or hamster spermatozoa (data not shown).
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We next tested for the presence of the endogenous sperm nuclease in humans. Human semen samples were collected from fertile donors and isolated by Percoll gradients. Osmolarity measurements for the 2 solutions used—HTF with 0.25% TX100 (262 mOsm) and HTF with 10 mM MgCl2 and 0.25% TX100 (284 mOsm)—did not vary significantly and were below the standard of 300 used in conventional intracytoplasmic sperm injection. As shown in Figure 6, fresh sperm samples incubated with TX100 at 37°C for 15 minutes digested their DNA to loop-sized fragments (Figure 6, lane 3). EDTA protected the cells from this activity. Spermatozoa with membranes that were not permeabilized by TX100 did not digest their DNA.
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However, the human sperm nuclease seemed to be activated by freeze/thawing, which was not the case for rodent spermatozoa. Earlier work has demonstrated that freeze/thawing can damage mouse sperm chromosomes if care is not taken to protect the DNA with EDTA and ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetra-acetic acid (EGTA) (Kusakabe et al, 2001). But this DNA damage was visualized (or observed) during zygote karyotyping and measured breaks in condensed sperm chromosomes. This type of DNA damage would result in fragments that are too large to assay by PFGE. In our experiments, mouse and hamster spermatozoa that were frozen overnight did not degrade their DNA into loop-sized fragments (data not shown). Therefore, in our initial experiments in humans, we used sperm samples that had been frozen and stored at -20°C before they were taken to our laboratory. We found that these samples often contained DNA that was already degraded to loop-sized fragments. To test whether freeze/thawing was the cause of this instability, 1 semen sample was separated into 2 aliquots: one that was used immediately after Percoll gradient separation (Figure 6, lanes 1-5), and the other that was frozen at -20°C overnight before treatment (Figure 6, lanes 6-10). The same sample that showed intact sperm DNA when used fresh (Figure 6, lane 1) had extensive DNA degradation when frozen, even when MgCl2 was not added (Figure 6, lane 6). EDTA was able to prevent this degradation in most cases (Figure 6, lane 10), which suggests that the human sperm nuclease was more easily activated than that of rodents and that DNA degradation resulting from unprotected freezing of human spermatozoa can be detected by PFGE.
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Discussion |
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Relationship of Sperm Chromatin Structure to the Sperm Nuclease— Mammalian sperm chromatin is so highly compact that it is resistant to biochemical and mechanical insults that destroy somatic cell DNA such as SDS (Ward et al, 1989), sonication (Tateno et al, 2000), and even high temperature (Yanagida et al, 1991). We have recently shown that protamine-bound sperm chromatin is much more resistant to DNAse I digestion than histone-bound DNA in somatic cells (Sotolongo et al, 2003). As described in the "Introduction," we proposed a donut loop model for sperm chromatin structure in which nuclease-sensitive regions of chromatin are interspersed between each toroid (Ward, 1993; Sotolongo et al, 2003). We demonstrated that these toroid linkers were also the sites of matrix attachment regions (MARs), the sites at which DNA loop domains are associated with the sperm nuclear matrix. In this report, we have shown that mouse and human sperm chromatin has a similar type of organization. The donut loop model therefore provides a structural framework for the action of the sperm nuclease we report here.
In testing our donut loop model in humans, we found that the sperm chromatin seemed to be more sensitive to exogenous DNAse I than either mouse or hamster spermatozoa. Sakkas et al (2002) reported that human spermatozoa treated with TX100 released small fragments of DNA with the use of both DNAse I and micrococcal nuclease. DNAse I released roughly 8-kb fragments from spermatozoa that had normal semen parameters, whereas samples with abnormal semen parameters released smaller fragments, down to 500 bp. Similar results were found with micrococcal nuclease. The apparent differences between that study and ours can be explained by the different techniques used to examine the DNA. We used PFGE, which separates DNA fragments from 2 mb to 2 kb, whereas Sakkas et al (2002) used conventional gel agarose. Our studies suggested that, given more time, the human sperm DNA was digested to smaller fragments, thus being consistent with the work of Sakkas et al (2002). Our study only used semen samples with normal parameters. Given the data of Sakkas et al (2002), we would predict that those samples with abnormal semen parameters would be more sensitive to DNAse I in generating the higher molecular fragments we report in this study.
The differences we found between hamster and mouse vs human spermatozoa regarding sensitivity to DNAse I cannot yet be explained. It is possible that this is because of a basic difference in the chromatin structure between the 3 species. Human spermatozoa have a larger percentage of histones than mouse or hamster (Tanphaichitr et al, 1978), but the ratio of protamine 1 to protamine 2 in humans (0.98; Balhorn et al, 1988) is in between that of mouse (0.46) and hamster (1.98; Zhang et al, 1996), so this is not the likely cause. The increased sensitivity might be related to the source: human sperm from ejaculates vs rodent spermatozoa from the epididymides. It is also possible that a contaminating nuclease from semen somehow gained access to the human samples because the latter were ejaculates. However, we view this as un-likely because the human samples we used were purified by Percoll gradients and thereby were separated from most of the seminal fluid components. As noted before, the spermatozoa were more than 90% motile after this step, and it is unlikely that the membranes were disrupted. Still, until the nuclease is clearly identified, we cannot rule out the possibility of a contaminant from the seminal fluid for the human spermatozoa.
Another cause of concern was the possibility that the changes in osmolarity that occurred because of the addition of the divalent cations might also activate a nuclease. We view this as unlikely because we were permeabilizing the membranes with TX100, so that changes in osmolarity will not affect the membranes. The changes within the solutions used for each species did not vary significantly (from 83 to 107 mOsm for the mouse and hamster and from 267 to 284 mOsm for the human). However, the difference in osmolarity between the HTF solutions used for human and the Tris solutions used for hamster and mouse was significant. It is possible that this played a role in the rapid activation of the human nuclease. We do not believe this to be the case because the human nuclear activity depended on membrane permeabilization. Two solutions with the same osmolarity (Figure 6, lanes 2 and 3) exhibited very different DNA digestion patterns on the basis of the presence or absence of TX100. However, we cannot rule out the possible effect of osmolarity on the nuclease activity at this time.
The endogenous sperm nuclease digests sperm chromatin to the same size as exogenous DNAse I, but we cannot as yet be certain that the endogenous sperm nucleases are cleaving the chromatin at the same sites as the exogenous DNAse I, and we are currently working on more molecular-based techniques to address this question directly. If the endogenous nuclease is digesting the sperm DNA at the MARs, it would share another similarity to the Ca/Mg-dependent nucleases that are activated in somatic cell apoptosis (see the next section).
Identity of the Sperm Nuclease—
The most immediate problem that our research now faces is to identify the
sperm nuclease whose activity we now report. Pittoggi et al
(1999) have shown that sperm
nuclei contain one or more endogenous nucleases that digest the histone-bound
chromatin first, and Wykes and Krawetz
(2003) have shown that the
distribution between histones and protamines within DNAse I-sensitive regions
is not uniform. These data suggest that histone-bound DNA is more sensitive to
nucleases. Our data show that MARs are digested. Together, these data suggest
that histone-bound DNA may be associated with the nuclear matrix. Clues to the
identity of the nuclease activity described in this report can also be found
in at least 3 enzymes that also cleave DNA into loop-sized fragments that are
well described in somatic cells. The first is DNAS1L3 or DNAse , a
chromatin-associated nuclease that has also been isolated from bull semen
(Yakovlev et al, 1999), making
it the most likely candidate. The sperm nuclease we have described here is
present in the sperm nucleus, fitting the criterion of chromatin association.
The second nuclease is DFF40, which is activated by proteolytic cleavage of
its binding protein DFF45 (Zhang et al,
1998). The third enzyme that causes a similar pattern of DNA
degradation in somatic cells is topoisomerase II (Topo II;
Lagarkova et al, 1995;
Li et al, 1999;
Solovyan et al, 2002). Topo II
makes transient double-stranded breaks in the DNA during its catalytic
unwinding activity, and this process can be interrupted, causing permanent
breaks. Topo II is localized at the bases of DNA loop domains, and when it is
activated in somatic cells during apoptosis, it cleaves DNA at the MARs
(Gromova et al, 1995;
Li et al, 1999). Razin and
colleagues have demonstrated that both the Ca/Mg-dependent nuclease and Topo
II cleave DNA at sites that are either identical or very near each other
(Gromova et al, 1995). This
suggests that the 2 different types of enzymes might play related roles in
somatic cell apoptosis.
Our data also indicated that sperm DNA is degraded completely in some cases (Figure 1); therefore, either the endogenous sperm nuclease we describe here is also capable of further digestion or another nuclease exists in spermatozoa that further degrades the sperm DNA. At least 1 nuclease, DNAS1L3, that digests somatic cell DNA into loop-sized fragments will digest naked DNA more completely when incubated for longer time periods with isolated nuclei (Yakovlev et al, 2000). Thus, the more complete digestion of sperm DNA is not unprecedented. We showed previously that protamine-bound DNA was less sensitive to DNAse I than histone-bound DNA (Sotolongo et al, 2003). However, the results reported here suggest that the sperm nuclease is capable of digesting protamine-bound DNA also, given enough time. It is worth noting that the DNAS1L3 only required 1 hour at 37°C to digest rat cerebellar nuclei to smaller fragments (Yakovlev et al, 2000), whereas the sperm nuclease required overnight digestion in the presence of both Mg2+ and Ca2+ in the hamster.
Although it is not yet clear what the function of this sperm nuclease is, the 3 enzymes described above digest chromatin into large, loop-sized fragments and act at the bases of the DNA loop domains. They or related sperm-specific proteins represent likely candidates to date. Cagan (2003) have recently demonstrated that Drosophila spermatids "borrow" the apoptotic molecular machinery to remove most of the cytoplasm in the last stages of spermiogenesis. This established a precedent for testing somatic apoptotic nucleases for an unknown function in spermatozoa.
Possible Reasons for the Existence of a Sperm Nuclease— The data we report here also bring to mind the question of the function for such nuclease activity in mature spermatozoa. Because the only other similar activity known is the degradation of DNA fragments during apoptosis, we must consider this function a possibility in spermatozoa. Several reports have described apoptotic-like events and apoptotic-related proteins in spermatozoa (Blanc-Layrac et al, 2000; Sakkas et al, 2002; Cayli et al, 2004; Martin et al, 2004), and if spermatozoa have an apoptotic mechanism, the nuclease we describe here might be part of it. The conclusion of an apoptotic-mediated mechanism is also supported by the work of Maione et al (1997), who showed that a process similar to apoptosis is triggered by exogenous DNA in sperm. Such a mechanism might assist macrophages in the female reproductive tract to degrade the majority of the spermatozoa that do not fertilize oocytes after mating. Also, it is possible that spermatozoa might respond to mutagenic agents or infection in the male or female reproductive tracts by cell death.
However at this point, we must keep open other possibilities as well, keeping in mind the paradigm for spermatogenesis to "borrow the apoptotic machinery" to accomplish the nonapoptotic functions described (Cagan, 2003). It is possible, for example, that the nuclease activity we report here by PFGE actually represents trapped cleavable complexes by Topo II. The SDS and proteinase K used to release the DNA into the gel would also digest the topoisomerase that holds the broken pieces of DNA together in vivo (Li et al, 1999). In this case, the double-stranded DNA breaks would represent an enzymatic intermediate of the Topo II-DNA complex that would normally be resolved. The function of such a topoisomerase in spermatozoa might be unwinding the chromatin during spermiogenesis during histone replacement or winding the chromatin during protamine replacement after fertilization.
Although its function and identity are still unknown, we have described the existence of an endogenous sperm nuclease in 3 mammalian species. The nuclease requires both Ca2+ and Mg2+ and digests the sperm chromatin into loop-sized fragments under certain conditions. Understanding this nuclease will have important implications not only for reproductive biology but also for the clinical manipulation of human spermatozoa.
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Footnotes |
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