Mol. Hum. Reprod. Advance Access originally published online on January 29, 2004
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Molecular Human Reproduction, Vol. 10, No. 3, pp. 203-209, 2004
© European Society of Human Reproduction and Embryology 2004
Structure of human sperm DNA and background damage, analysed by in situ enzymatic treatment and digital image analysis
1Sección de Genética y Unidad de Investigación, Hospital ‘Teresa Herrera’, Complejo Hospitalario Universitario Juan Canalejo, 15006 La Coruña, 2Unidad de la Mujer, La Coruña and 3Unidad de Genética, Facultad de Biología, Universidad Autónoma de Madrid, Spain
4 To whom correspondence should be addressed at: Sección de Genética, Hospital ‘Teresa Herrera’, Complejo Hospitalario Universitario Juan Canalejo, 15006 La Coruña, Spain. e-mail: JLFernandez{at}canalejo.org
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DNA breakage detection–fluorescence in situ hybridization (DBD–FISH) is a procedure to detect and quantify DNA breaks in situ, on a cell-by-cell basis. A comparison between sperm nuclei versus peripheral blood leukocytes using this method demonstrated that the nucleoids from mature human sperm are 12.7 times more sensitive to alkaline denaturation than those from human peripheral blood leukocytes. To investigate the origin of this alkali sensitivity, different approaches were employed. First, free 3'-OH ends of background DNA breaks were labelled by Klenow polymerase, or by DNA polymerase I following the in situ nick translation assay. Second, the presence of abasic sites, the other recognized DNA lesions that lends to constitutive alkali sensitivity, and DNA breaks with blocked 3' ends, were determined by in situ exonuclease III digestion prior to the polymerase labelling. The results demonstrated that the sperm nucleoid contains
2.5-fold higher density of background DNA breaks with 3'-OH ends, and also 2.8-fold higher density of basal abasic sites and DNA breaks with blocked 3' termini, than leukocytes. These differences only partially explain the significant alkali sensitivity of sperm DNA. However, in situ digestion with mung bean nuclease before DNA break labelling showed that sperm DNA is 9-fold more enriched in segments of ssDNA than DNA from leukocytes. The high frequency of partially denatured regions may result from a greater torsional stress of DNA loops in sperm chromatin due to its higher degree of compaction. Moreover, these short unpaired ssDNA stretches should be included in the category of alkali-labile sites detected by all techniques that measure DNA breaks through an alkaline unwinding step. These results provide new insights into the nature of DNA packaging in sperm nuclei. Key words: Key words: alkali-labile sites/alkaline DNA unwinding/chromatin structure/DBD–FISH/sperm DNA
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The integrity of sperm DNA is of crucial importance for balanced transmission of genetic information to future generations. There is good evidence that sperm DNA damage may lead to conception failure, abortions, malformations, and genetic diseases. Furthermore, the origin of 80% of de novo structural chromosome aberrations in man are of paternal origin (Tomar et al., 1984). During the last two decades, an interspecies IVF system between human sperm and golden hamster oocytes allowed the study of sperm-derived chromosomes. These studies demonstrated that human sperm contain a higher incidence of baseline numerical and structural chromosome aberrations than somatic cells, as well as a higher incidence of chromosome aberrations after in vitro and in vivo exposure to different mutagens (Martin et al., 1989; Genesca et al., 1990; Kamiguchi and Tateno, 2002). More recently, the use of fluorescence in situ hybridization (FISH) demonstrated a significantly higher incidence of structural aberrations than numerical abnormalities in human sperm, confirming the high frequency previously measured by the hamster oocyte method (Sloter et al., 2000). Moreover, it is now recognized in humans that couples in whom the male semen contains a high percentage of sperm cells with heavily fragmented DNA have a very low potential for natural or assisted reproduction. Thus, recent clinical studies indicate that DNA fragmentation levels >30%, as measured by the sperm chromatin structure assay (SCSA) test, are incompatible with the initiation and maintenance of a term pregnancy (Evenson et al., 1999; Larson et al., 2000).
At the DNA level, different techniques have been applied to sperm cells to analyse DNA damage. Many of them are based on the detection of DNA strand breaks through an alkaline unwinding treatment. The alkali transforms DNA breaks into ssDNA that are initiated from the ends of the break (Rydberg, 1975). These techniques differ in the ways in which the ssDNA is analysed. The alkaline single-cell gel electrophoresis (SCGE) is a well-established morphological procedure that allows cell-by-cell evaluation. In this method, the length and amount of DNA that migrates to the tail, as determined after DNA staining with a fluorochrome, is proportional to the amount of breaks per cell. Alkaline unwinding detects alkali-labile sites in addition to single- and double-DNA strand breaks, i.e. DNA lesions or modifications that are transformed into DNA breaks by the alkali. These DNA modifications are classically referred to as DNA abasic sites, apurinic or apyrimidinic (AP), or deoxyribose lesions (von Sonntag, 1987). Though a steady-state level of DNA breaks and AP sites spontaneously occurs in living cells (Atamna et al., 2000), deoxyribose lesions are probably more characteristic of radical or exogenous damage. Surprisingly, using the SCGE assay, it was found that DNA from mature sperm cells contains a large number of alkali-labile sites (Singh et al., 1989; Haines et al., 1998). Alkaline elution techniques also supported these results (Singh et al., 1989; Van Loon et al., 1991, 1993). The nature of these alkali-labile sites has not been defined, although it has been speculated that they may be a characteristic of condensed chromatin. This assumption is supported by the fact that chicken erythrocytes and mouse kidney DNA, both highly condensed, revealed a similar high density of constitutive alkali-labile sites (Singh et al., 1989; Fairbain et al., 1994).
In the present report we have applied a battery of in situ DNA labelling techniques to gain information on the nature of the intense background sensitivity to alkali of DNA from human sperm cells, as well as to establish baseline DNA damage, in comparison with somatic cells (i.e. human blood leukocytes).
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Preparation of nucleoids in agarose microgels
The study was carried out with human blood leukocytes and mature sperm from the same donor, in two healthy donors, obtained with informed consent. Sperm cells in raw semen were diluted in Roswell Park Memorial Institute 1640 medium (Gibco, Invitrogen, UK) to obtain a sperm concentration of 10x106/ml. Leukocytes in buffy coat and diluted sperm cells were separately mixed with low-melting point agarose (Pronadisa; Hispanlab, Spain) to produce a final concentration of 0.7% agarose at 37°C. Then 28 µl of each mixture was pipetted onto the same glass slide precoated with 0.65% standard agarose dried at 80°C, covered with a cover slip (12x60 mm), and left to solidify at 4°C. The longitudinal half of the slide contained the leukocytes, whereas the other half contained the sperm. Thus, both cell types were simultaneously processed in the same glass slide, under the same conditions. Cover slips were carefully removed, and the slides immediately immersed horizontally in a tray with a large volume of lysing solution 1 [0.4 mol/l Tris, 0.8 mol/l dithiothreitol (DTT), 1% sodium dodecyl sulphate (SDS), 50 mmol/l EDTA, pH 7.5] for 10 min at room temperature, followed by incubation in lysing solution 2 (0.4 mol/l Tris, 2 mol/l NaCl, 1% SDS, pH 7.5) for 5 min at room temperature. Finally, the slides were thoroughly washed in phosphate-buffered saline (PBS), four times, 5 min each.
DBD–FISH procedure to detect DNA breaks and alkali-labile sites
After washing in PBS (Sigma, USA), the nucleoids were washed twice in 0.9% NaCl, 5 min each, and then incubated in an alkaline unwinding solution (0.03 mol/l NaOH, 1 mol/l NaCl), for 2.5 min at 7°C in the dark. This transforms DNA breaks and alkali-labile sites into short ssDNA regions. After neutralization with a 5 min incubation in 0.4 mol/l Tris–HCl (pH 7.5), the slides were washed in TBE buffer (0.09 mol/l Tris–borate, 0.002 mol/l EDTA, pH 7.5) for 2 min, dehydrated in sequential 70, 90 and 100% ethanol baths, 2 min each, and air-dried. A human whole genome probe (4.3 ng/µl in 50% formamide/2xSSC, 10% dextran sulphate, 100 mmol/l calcium phosphate, pH 7.0) (1xSSC is 0.015 mol/l Na citrate, 0.15 mol/l NaCl, pH 7.0), biotin-labelled by nick-translation, was denatured and hybridized overnight at room temperature on dried slides. After hybridization, slides were washed twice in 50% formamide/2xSSC, pH 7.0, for 5 min, and twice in 2xSSC pH 7.0, for 3 min, at room temperature. Hybridized probe was detected with streptavidin–Cy3 (1:200) (Sigma, USA) and cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI) in Vectashield (Vector, USA) (Fernández et al., 1998, 2002; Fernández and Gosálvez, 2002).
Labelling of DNA breaks with 3'-OH ends
Two methods were used to label DNA breaks with 3'-OH ends.
End labelling with Klenow polymerase.
After the PBS washes, the slides were incubated four times, 5 min each, in excess reaction buffer for Klenow polymerase (10 mmol/l Tris–HCl, 5 mmol/l MgCl2, 7.5 mmol/l DTT, pH 7.5). Reaction buffer (100 µl) containing 25 IU of Klenow polymerase (New England BioLabs, USA) and biotin-16-dUTP in the nucleotide mix were pipetted onto the slide, covered with a plastic cover slip, and incubated in a moist chamber for 30 min at 37°C. After washing in TBE buffer, the slides were dehydrated in sequential 70, 90 and 100% ethanol baths, and air-dried. The incorporated biotin-16-dUTP was detected by a 30 min incubation with streptavidin–Cy3, and slides were DAPI-counterstained. Reaction buffer only was placed on one area of the slide, as a control. The microgel between the areas with and without the polymerase was scratched to avoid the possible diffusion of the enzyme to the control area.
In situ nick translation (ISNT) labelling with DNA polymerase I.
The protocol and reaction buffer were similar to that of Klenow polymerase. In this case, 20 IU of DNA polymerase I (New England BioLabs) in 100 µl of reaction buffer (10 mmol/l Tris–HCl, 5 mmol/l MgCl2, 7.5 mmol/l DTT, pH 7.5) were incubated on the slide.
Labelling of AP sites and DNA breaks with blocked 3' ends
After PBS washes, the nucleoids were incubated in excess exonuclease III reaction buffer (0.66 mmol/l MgCl2, 66 mmol/l Tris–HCl, pH 7.0), four times, 5 min each. A transverse half of the slide, including an area of the microgel containing sperm cells and another area of the microgel containing leukocytes, were incubated with 100 IU of exonuclease III (New England BioLabs) in 50 µl of reaction buffer, for 30 min at 37°C. The other half was covered by exonuclease III buffer alone. Previously, the microgel between both halves had been scratched. After four thorough washings in PBS, the slides were processed for Klenow end labelling or ISNT with DNA polymerase I. as described above. The increase in labelling within the exonuclease III-digested area compared to the undigested area, (exonuclease III + Klenow or DNA polymerase I) – (Klenow or DNA polymerase I alone), is dependent on AP sites cleaved by the nuclease as well as on possible DNA breaks with blocked 3' ends.
In another set of experiments, after exonuclease III digestion, the slides were washed in TBE buffer, dehydrated in ethanol baths and air-dried. The ssDNA generated by the nuclease from DNA breaks and AP sites was detected by hybridization with the whole genome probe, as described before. This is equivalent to FISH with an enzymatic denaturation (Gosálvez et al., 2002).
ssDNA labelling
The protocol was similar to that of the AP site labelling. In this case, initial incubations were in excess mung bean nuclease reaction buffer (50 mmol/l Na acetate, 100 mmol/l NaCl, 1 mmol/l ZnCl2, pH 5), followed by 25 IU of mung bean nuclease (New England BioLabs) in 50 µl of reaction buffer, for 45 min at 37°C. Finally, the slides were processed for Klenow end labelling. The increase in labelling within the mung bean nuclease-digested area compared to the undigested area, (mung bean + Klenow) – (Klenow alone), is dependent on ssDNA segments cleaved and removed by the nuclease.
Fluorescence microscopy and digital image analysis
Slides were viewed under a DMRB epifluorescence microscope (Leica, Germany) equipped with a DMRD photoexposer, PL Fluotar x100 objective and appropriate fluorescence filters for DAPI and Cy3. Cy3 Images were acquired using a high sensitivity CCD camera (Ultrapix 1600; Astrocam) that detects >16 000 grey levels and allows subtraction of the current dark image and correction for non-uniform sample illumination. Groups of 50 digital images, one per cell, were taken for each experimental point, stored in the file format of the camera (.apf) and then converted to .img files. Exposure times were different depending on the experiment, in order to avoid saturation of the signal. Thus, DBD–FISH images were taken for 200 ms, images from DNA break labelling with Klenow and ISNT, as well as from exonuclease III-treated nucleoids, were captured for 1 s, and those from the experiment with mung bean nuclease were exposed for 500 ms. Each experiment was repeated at least twice. Image analysis was performed using a semi-automatic routine designed with Visilog 5.1 software (Noesis, France). This allows for thresholding, background subtraction, and measures the whole fluorescence intensity [surface area (in pixels)xmean fluorescence intensity (in arbitrary grey levels)] of the signal. Statistical analysis was carried out using Student’s t-test. It should be noted that, for comparison with leukocytes, signals from sperm were multiplied by 2 to correct for the difference in DNA ploidy.
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DBD–FISH is a sensitive technique to detect and quantify DNA breaks in single cells either in the whole genome or within specific DNA sequence areas. Cells trapped in an inert agarose microgel over a slide are lysed and immersed in an alkaline unwinding solution that produces ssDNA motifs starting from the ends of the DNA breaks. After dehydration, the nucleoids are incubated with DNA probes. The amount of hybridized probe in a target is related to the amount of melted ssDNA generated by the alkali, which is in turn proportional to the degree of local DNA breakage (Fernández and Gosálvez, 2002). Using a whole genome probe, we had previously observed that nucleoids from mature human sperm yielded very strong fluorescent signals (Fernández et al., 2000). Figure 1a and b presents DBD–FISH signals from human blood leukocytes and mature sperm cells from the same individual, processed in the same slide under the same experimental conditions. Using a restrictive alkaline incubation (2.5 min at 7°C), both cell types exhibit a heterogeneous labelling of the nucleoids, showing areas with different signal strengths. A quantitative analysis of the fluorescence (Table I) reveals that under these conditions, and correcting for the DNA content, the sperm cells have 12.7-fold more fluorescent signal than that from leukocytes. This extreme alkali sensitivity of DNA from sperm cells had also been reported using the alkaline SCGE or alkaline elution assays (Singh et al., 1989; Van Loon et al., 1991, 1993). Unlike those sperm cells with heavily fragmented DNA, this result was only obtained when the alkaline unwinding step was performed on previously deproteinized cells (Fernández et al., 2000), suggesting the presence of a high density of constitutive alkali-labile sites in the sperm DNA, that were masked by the higher-order chromatin organization with protamines. Otherwise, DTT could generate sulphur-centred radicals on the protamines, and not on histones, due to the disruption of disulphide bridges. This could potentially originate damage in the close DNA molecule, specifically in sperm nucleoids. Nevertheless, additional experiments using either proteinase K (1 mg/ml, overnight, at 37°C) instead of DTT, or using the lysing solutions complemented with freshly added 10% dimethylsulphoxide as radical scavenger, showed the same differential result between sperm cells and leukocytes, after DBD–FISH. It discards an artefact created by the cell preparation technique. The nature of these constitutive alkali-labile sites has not been established. Besides masked DNA breaks, they may correspond to AP sites, both turning into ssDNA by alkaline treatment (Téoule, 1987; von Sonntag, 1987). In fact, DNA breaks and AP sites are known to be at a steady-state level in living cells, continuously being generated and repaired.
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Different in situ labelling protocols were performed to study the nature of the strong alkali-labile sensitivity of human sperm DNA. These protocols assayed for DNA breaks or AP sites. Background DNA breaks with 3'-OH ends were labelled using both Klenow and DNA polymerase I (Dierendonck, 2002; Wood, 2002). The Klenow fragment of Escherichia coli DNA polymerase I is routinely employed for in situ labelling of DNA breaks. This enzyme catalyses the addition of nucleotides to the 3'-OH end of a DNA break using the complementary DNA strand as template. Though the 5'–3' exonuclease activity from DNA polymerase I is absent, the Klenow fragment conserves its 3'–5' exonuclease proofreading activity, which could produce a nick translation effect. Nevertheless, in the presence of dNTP the polymerase activity counteracts further degradation, so the nucleotide at the 3'-OH end will be continuously replaced. The final result is the gap or end-filling, and subsequent labelling by the incorporation of biotin-16-dUTP. Using this approach, sperm nucleoids exhibit 2.6-fold more signal than leukocytes (Figure 1c, d; Table I). That is, the density of basal single-strand breaks, nicks or gaps with 3'-OH ends, appears to be 2.6-fold higher in sperm than in leukocytes. In the case of DNA polymerase I, in addition to the 5'–3' polymerase activity and the 3'-5' exonuclease activity, the enzyme has a concurrent 5'–3' exonuclease action. Therefore, the primer strand is hydrolysed at the 5' side of the nick while the synthetic activity catalyses the addition of nucleotides to the 3' side. The simultaneous hydrolysis and synthesis results in the translation of the nick along the DNA duplex in the 5'–3'direction, thus resulting in the incorporation of a great amount of biotin-16-dUTP molecules per single DNA strand break. This is reflected in a higher background labelling of DNA breaks in both leukocytes and sperm with the DNA polymerase I than when using the Klenow fragment (Figure 1e, f). Nevertheless, the difference in signal between both cell types is very close to that found for Klenow polymerase, that is, sperm cells contain 2.3 higher density of background DNA breaks than leukocytes (Table I). It is accepted that basal DNA breaks in the living cell correspond to single-strand breaks or nicks, double-strand breaks being extremely rare, unless apoptotic degradation or damage is induced by exogenous agents. The labelling systems we employed require that the DNA break should contain a free 3'-OH end, as in the terminal uridine nucleotide end labelling (TUNEL) assay, using the terminal deoxynucleotidyl transferase (TdT). Though the latter enzyme is primer and template independent, this is only of interest in the case of the necessity for labelling blunt-ended or 5'-recessed fragments, that cannot be detected by the polymerases (Walker et al., 2002). Nevertheless, this only should be the case when analysing double-strand breaks, which are practically absent in living cells.
Free 3'-OH ends are typical of the transient DNA breaks generated by the excision repair machinery to correct for spontaneous base damage and AP sites in the living cell. Though 3'-OH ends may be more predominant in background damage, 3'-ends with phosphate or phosphoglycolate groups could be present in other single-strand breaks. These seem to be a consequence of oxidative damage, and behave as blocks to the polymerase activity (Téoule, 1987; von Sonntag, 1987). Other DNA lesions present under physiological conditions that may lead to alkali sensitivity are AP sites. These modifications are generated by spontaneous base loss through cleavage of the glycosyl bond between deoxyribose and purines or pyrimidines. The alkali incises the DNA 3' to the AP site, transforming it into a single-strand break. It has been established that the spontaneous depyrimidination rate seems to be 20 times slower than that of depurination (Lindahl and Karlström, 1973). Though with high variability depending on the reports, the steady-state level of AP sites in living cells has been recently estimated as <1 AP site per 106 nucleotides, and may increase with ageing (Atamna et al., 2000). To investigate other DNA alterations, such as presence of AP sites under physiological conditions, that may lead to alkali sensitivity, we used a different experimental approach based on E. coli exonuclease III activity. Exonuclease III is a 3'–5' exonuclease specific for double-stranded DNA, which removes nucleotides starting from 3'–ends, producing ssDNA patches. These 3' ends may contain either a free hydroxyl group or modifications that block the polymerases, since exonuclease III removes the 3' blocking ends, to form free 3'-OH groups, suitable substrates for DNA polymerases. This enzyme also has AP endonuclease activity, so that a 3'-OH end is specifically generated after in situ treatment of DNA. 3'-OH ends generated by exonuclease III can be labelled by the Klenow fragment or by ISNT with DNA polymerase I (Liu et al., 2002). This is a similar approach to that carried out by Legault et al. (1997), but they employed T4 DNA polymerase to label free 3'-OH ends, whereas the sum of AP sites and single-strand breaks with both free 3'-OH and 3' blocking groups were labelled using E. coli endonuclease IV previously to the polymerase, instead of exonuclease III. Nucleoids preincubated with exonuclease III show the expected increase in the signal due to the extra labelling of AP sites and DNA breaks with blocked 3' termini as compared to nucleoids treated with either Klenow or DNA polymerase I to label DNA breaks (compare Figure 1c–f with g–j). Thus, the contribution of AP sites and these kinds of DNA breaks is estimated by subtracting the value of the polymerase signal from the exonuclease III–polymerase signal. In the case of the Klenow labelling, DNA from sperm cells exhibits 3.1-fold higher density of AP sites and DNA breaks with 3' blocking groups than that from leukocytes (Figure 1g, h; Table I); whereas after ISNT this difference is 2.6-fold (Figure 1i, j; Table I). Otherwise, the ssDNA generated by exonuclease III from both the DNA breaks and the cleaved AP sites can be detected by incubation with a whole genome probe (Figure 1k, l). This is the equivalent to a FISH using an enzymatic denaturation (Gosálvez et al., 2002). The quantitative analysis showed that sperm cells have 4.5-fold more signal from background DNA breaks, with 3'-OH and blocked 3' termini, and AP sites together, than leukocytes. It is higher than the difference detected when these whole lesions were labelled with exonuclease III–Klenow or exonuclease III–DNA polymerase I (Table I). This could be explained because sperm nucleoids show internal areas of strong hybridization of the whole genome probe after exonuclease III–FISH, far more marked than in leukocytes (Figure 1k, l). These areas may correspond to chromocentres of clustered centromeric–pericentromeric repetitive satellite DNA sequences (Zalensky et al., 1995), which hybridize more strongly when using a whole genome probe (Fernández et al., 2000).
Altogether, taking into account the differences in genome size, sperm DNA contains 2.5-fold more background single-strand breaks with free 3'-OH ends and a close magnitude of extra AP sites and possible DNA breaks with 3' blocking groups, than leukocyte DNA. Besides being physiological, there is the possibility of generation of some of these lesions in vitro, during the technical processing. Nevertheless, working on an inert agarose matrix minimizes this problem, stabilizing the relaxed DNA loops, avoiding both chemical oxidation during phenol extraction and physical shearing (Legault et al., 1997), being the base of the SCGE and DBD–FISH assays to detect induced DNA breaks. Moreover, there should be a similar sensitivity of nucleoids from sperm or leukocytes, to spontaneously produced DNA lesions during the incubations, so that the observed differences cannot be attributed to them. Both DNA breaks and AP sites exist in a steady-state in living leukocytes, being continuously produced and repaired. Nevertheless, the mature sperm lacks DNA repair capacity (Robbins, 1996), and appears highly vulnerable to radiation and some chemicals or their metabolites (Kamiguchi and Tateno, 2002). Therefore, DNA damage could accumulate as sperm age and/or are in contact with potential exogenous damaging agents. Thus, the DNA repair activity of the penetrated oocyte should be of enormous importance to correct for the background damage of human sperm DNA (Genesca et al., 1992).
According to the DBD–FISH results where an extreme alkali-sensitivity of naked sperm DNA was evident in sperm cells compared to leukocytes, the relatively higher density of single-strand breaks and AP sites is insufficient to fully explain the situation. It is evident that these spontaneous DNA lesions should not be the only modifications that confer alkali sensitivity in undamaged cells. It has been stated that DNA in chromatin from mitotic cells is more sensitive to denaturation than is the DNA in chromatin of interphase cells (Darzynkiewicz et al., 1987; Darzynkiewicz, 1994). The ssDNA was detected by flow cytometry after acridine orange staining. The same group also demonstrated that this sensitivity was related to a higher abundance in mitotic chromosomes of DNA segments sensitive to single strand-specific nucleases, such us S1 and mung bean nucleases (Juan et al., 1996). Based on these observations, it was hypothesized that the stronger torsional tension in DNA loops of mitotic chromosomes may lead to the formation of a higher frequency of single-stranded hairpin-loop structures which are sensitive to single strand-specific nucleases. These localized unpaired DNA segments may be too small to be detectable by Acridine Orange staining or by FISH. Nevertheless, they would behave as initiation sites from which the denaturing agents would initiate the generation of extensive ssDNA that becomes detectable by these procedures.
The chromatin of mature mammalian sperm is specifically arranged in an extremely compact and stable organization. Based on the doughnut loop model, the protamine-bound DNA is coiled into loops that collapse into a toroid of tightly packed chromatin. Each toroid would represent a single DNA loop domain of 40–50 kb, attached at its base to the nuclear matrix (Allen et al., 1997; Ward, 1997). As a result, when chromatin is organized in a protamine complex, it becomes six times more condensed than in metaphase chromosomes (Ward and Coffey, 1991). This organization is stabilized by inter- and intramolecular disulphide bonds in protamines, resulting in a very rigid, crystalline-like structure (Balhorn, 1982). Moreover, the DNA loops from sperm chromatin are smaller than those from somatic cells, the latter with an average size of 90 kb, or even longer, so their relative density should be higher in sperm cells (Nadel et al., 1995; Klaus et al., 2001). In the case of somatic cells, S1-hypersensitive sites have been mapped close to the matrix attachment regions (MAR), located at the base of the DNA loops (Targa et al., 1994). MAR contain base-unpairing regions (BUR) that may ligate specific nuclear proteins which could be involved in the establishment of the tissue-specific nuclear architecture and regulation (Shanon, 2003; Cai et al., 2003). These facts imply that the presence of localized unwound sites by severe topological tension should be far more frequent in the chromatin from mature sperm cells. We have compared the in situ end labelling of the DNA breaks generated by mung bean nuclease in nucleoids from sperm and leukocytes (Figure 1m–p). Mung bean nuclease was chosen because, unlike S1 nuclease, this enzyme will not cleave the DNA strand opposite a nick, so it is more specific of ssDNA. After subtraction of the signal corresponding to Klenow labelling alone, i.e. the component due to background DNA breaks, it was found that the nucleoids of sperm cells contain 9-fold higher density of short ssDNA stretches than the nucleoids from leukocytes (Figure 1 m–p; Table I). This supports the great abundance of partially denatured ssDNA segments as the main cause of the strong alkali sensitivity of sperm DNA. In the case of histone-organized chromatin, these ssDNA segments had been detected by nuclease digestion, without previous protein extraction (Juan et al., 1996). In our case, it is difficult to establish their presence in intact sperm nuclei, since their compaction level restricts the accessibility of nucleases in comparison with histone-organized chromatin, and it should be borne in mind that the alkali sensitivity of sperm DNA was only detected after protein removal. Nevertheless, it is interesting to note that the DNA segment sensitive to S1 nuclease in organized chromatin is also sensitive as naked DNA, when it is stressed in recombinant DNA plasmids (Larsen and Weintraub, 1982). In any case, besides AP sites, the presence of partially denatured DNA segments should be considered as genuine alkali-labile sites, either spontaneous or induced by DNA damaging agents, and be taken into consideration in all the procedures that employ an alkaline denaturation to detect and quantify DNA breakage. Finally, since ssDNA is known to be much more sensitive than double-stranded DNA to several types of DNA damage (Shapiro, 1981; Legault et al., 1997), the higher density of unpaired DNA segments could explain the relatively higher frequency of background DNA damage and sensitivity to several mutagens, in mature sperm. It may be useful to evaluate the behaviour of germ cells from different stages of maturation, using our experimental approach. Since remarkable changes in chromatin structure and organization are apparent, this could result in different responses to the unwinding treatment.
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We are very grateful to Barbara Hamkalo (Department of Molecular Biology and Biochemistry, University of California, Irvine) for the critical reading of the manuscript. This work was supported by the Fondo de Investigaciones Sanitarias (FIS 01-3113), Plan Gallego de Investigación y Desarrollo Tecnológico de la Xunta de Galicia (PGIDIT-01BIO02E) and PGIDIT 02PXIC91603PN, and the Consejo de Seguridad Nuclear from Spain.
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Submitted on September 4, 2003; resubmitted on November 18, 2003; accepted on November 20, 2003.
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