DNA topoisomerases I and II in human mature sperm cells: characterization and unique properties
1 IVF unit, Department of Obstetrics and Gynaecology, Soroka University Medical Center, Faculty of Health Sciences, Ben-Gurion University, Beer-Sheva 84105, Israel 2 Shraga Segal Department of Microbiology and Immunology, Faculty of Health Sciences, BGU Cancer Research Center, Ben-Gurion University, Beer-Sheva 84105, Israel
3 Correspondence address. Tel: +972-8-6479537; Fax: +972-8-6479579; E-mail: priel{at}bgu.ac.il
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BACKGROUND: The condensed state of the human sperm's chromatin is essential for the compact structure of the spermatozoon head, which is important for the fertilization process. The enzymes DNA topoisomerases (topo I and topo II) are responsible for the topological structure of the chromatin in somatic cells. The activities and the characterization of topoisomerases in mature human sperm cells have not been previously investigated.
METHODS: Sperm cells were purified from the semen of healthy donors by standard procedures and assays measuring the activities, protein size and sensitivity to inhibitors of topoisomerases were performed.
RESULTS: Topo I and topo II DNA relaxation activities are present in nuclear extracts derived from human sperm. The sperm topo I activity is inhibited by camptothecin, similarly to the somatic enzyme. An 85 kDa sperm protein, compared with the 100 kDa somatic topo IB enzyme, reacted with anti-human topo I antibody. Sperm topo II lacks the DNA decatenation activity of the somatic enzyme and a 97 kDa protein, compared with the 170 kDa somatic topo II
enzyme, was detected with anti-human topo II antibody. Sperm nuclear extracts contained inhibitors of somatic topo II decatenation activity.
CONCLUSIONS: Topoisomerase I and II activities as well as topo I and topo II proteins are present in mature human sperm cells. These enzymes possess unique properties compared with their somatic counterparts.
Key words: decatenation/DNA relaxation/human sperm/topoisomerase I/topoisomerase II
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Chromosomes in terminally differentiated mammalian spermatozoa are extensively condensed by protamines (Gatewood et al., 1987
, 1990; Hecht, 1990; Gardiner-Garden et al., 1998). Unlike somatic chromatin, only a small proportion of the chromatin is in the structure of nucleosomes (Gatewood et al., 1987). The condensation of the sperm chromatin is believed to be essential for male fertility. Men with normal semen parameters exhibit a condensed chromatin, whereas spermatozoa with low motility are more likely to contain loosely packaged chromatin (Olivia, 2006; Mantas et al., 2007). Therefore, one may assume that the condensation state of the sperm chromatin should be maintained by a tight regulation of enzymes that are responsible for the topological state of the DNA, namely DNA topoisomerases. These are ubiquitous nuclear enzymes, which are involved in the condensation and decondensation processes of the chromatin. They participate in most of the DNA transactions such as: replication, transcription, recombination, nucleosomal assembly and chromosome segregation during cell division. Topoisomerases are classified as type I or type II, and members of each family of enzymes are distinct in sequence, structure and function (Wang et al., 1997; Wang, 2002; Corbett and Berger, 2004). The mammalian topo I (topo IB) is present in virtually all somatic cells and is a 100 kDa polypeptide encoded by a single copy gene (Stewart et al., 1996). Topo IB can relax both positive and negative supercoiled DNA by the formation of a transient single strand DNA break in which the active site tyrosine becomes attached to the 3' phosphate end of the cleaved strand followed by the rotation of the DNA and religation process (Krogh and Shuman, 2000; Champoux, 2001; Wang, 2002; Leppard and Champoux, 2005). The topo I inhibitor camptothecin (CPT) is a potent anti-cancer agent and its derivatives topotecan (TPT) and irinotecan (CPT-11) are used today for the treatment of ovarian cancer and colorectal cancer, respectively (Pommier, 1998, 2006; Pommier et al., 1999; Mathijssen et al., 2002). Topo II is essential in all living cells since they are the only enzymes that can decatenate or unlink intact DNA duplexes; they participate in the condensation and segregation of chromosomes, determining both DNA secondary and tertiary structures. Accordingly, they are absolutely required at the stage of chromosome segregation. Mammalian cells express two topo II isoforms, designated as (170 kDa protein) and 
(180 kDa protein), that are encoded by different genes (Forterre et al., 2007). Topo II relaxes supercoiled DNA by introducing double strand DNA breaks and an ATP dependent transport of one DNA duplex through another (Corbett and Berger, 2004). Topo II is a specific target for some of the most active anti-cancer drugs used in the treatments of human malignancies (Martincic and Hande, 2005). Among the topo II-targeted agents currently in clinical use is etoposide (VP-16) (Baldwin and Osheroff, 2005).
The involvement of topo II in spermatogenesis in mammals has been previously suggested (Galande and Muniyappa, 1996; Bakshi et al., 2001; Laberge and Boissonneault, 2005a,b). It was shown that topo II is differentially expressed during the development of the post-natal testis and that testosterone regulates the expression of topo II in prepubertal animals (Bakshi et al., 2001). Topo II activity was described in Xenopus laevis spermatocytes and pre-elongated spermatids and the presence of this enzyme in nuclei of cells at the different spermatogenic stages was demonstrated (Morse-Gaudio and Risley, 1994). In addition, a truncated topo II protein was observed in mouse sperm, but the activity of this enzyme was not examined (St Pierre et al., 2002).
The highly condensed chromatin in mature spermatozoa suggests that the activity of topoisomerases in these cells (if present) should be tightly regulated in order to maintain this essential condensed structure.
The activity and regulation of topoisomerases in mature human sperm has not been previously investigated. We, therefore, examined the presence of the activities of topoisomerases and characterized the properties of these enzymes in human mature sperm cells.
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Sperm collection and isolation
The present research involved human subjects and was approved by the Institutional Helsinki committee.
Human sperm cells were isolated from fresh semen obtained from fertile donors. Sperm parameters (count, percent of motile sperm and quality of motility) were evaluated by using a Makler counting chamber (Makler, 1978). The samples that showed normal sperm parameters and contained 50–100 x 106 sperm/ml were used. Samples were washed of semen by dilution 1:1 with PBS (0.136 M NaCl, 27 mM KCl, 18 mM KH2PO4, 0.1 M Na2HPO4) and centrifugation at 300 g for 10 min. The sperm cells were separated from leucocytes and other accompanying cells using a standard Hypaque-Ficoll (Sigma-Aldrich, Rehovot, Israel) separation assay. Briefly, the sperm pellet was resuspended in 1 ml PBS and was carefully placed over Ficoll gradient then centrifuged at 250 g for 25 min. The sperm pellet was resuspended and washed in PBS. Sperm pellets from three donors were pooled and nuclear protein extracts were prepared.
Preparation of nuclear cell extracts
Nuclear extracts from sperm and from a human lymphoblastoid cell line, as somatic control cells, were prepared as previously described (Auer et al., 1982; Sambrook et al., 1989). Briefly, the sperm pellet was washed three times from Ficoll residues by resuspension of the cells in PBS followed by centrifugation as described earlier. The human lymphoblastoid cells (107 cells) were collected by centrifugation from the culture medium, and the cell pellet was washed three times with PBS. The cells (sperm or lymphoblastoid) were resuspended in hypotonic buffer A (10 mM Tris–HCl, pH 7.4, 10 mM NaCl, 1 mM EDTA and a mixture of protease inhibitors: 2 µg/ml aprotinin, 2 µg/ml leupeptin, 1 µg/ml pepstatin A, 2 µg/ml antipain, 100 µg/ml PMSF-phenylmethylsulfonyl fluoride; all were purchases from Sigma-Aldrich, Rehovot, Israel) and incubated in ice for 15 min. Cell lysis was performed by passing the solution 10 times through a 21G needle followed by centrifugation at 13 000 rpm at 4°C for 10 min. The supernatant was collected as the cytosolic fraction and the pellet containing intact nuclei was resuspended with buffer B (10 mM Tris–HCl, pH 7.4, 10 mM NaCl and 1.5 mM MgCl2 and protease inhibitors) and 1 M (final concentration) of NaCl was added for a 30 min incubation on ice. The samples were centrifuged (13 000 rpm, 4°C, 10 min), and the supernatant was collected as the nuclear extract. Total protein concentration was determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories, CA, USA).
Topo I assay
Increasing (or, alternatively, specific) concentrations of nuclear or cytoplasmic proteins were added to a topo I reaction mixture containing, at a final volume of 25 µl: 20 mM Tris–HCl (pH 8.1), 1 mM dithiothreitol (DTT), 20 mM KCl, 10 mM MgCl2, 1 mM EDTA, 30 µg/ml bovine serum albumin and 250 ng pUC19 supercoiled DNA plasmid (MBI, Fermentas, Hanover, MD, USA). Where indicated, topo I or topo II inhibitors were added to the reaction mixture. Following incubation at 37°C for 30 min, the reaction was terminated by adding 5 µl of stopping buffer [final concentration: 1% sodium dodecyl sulfate (SDS), 15% glycerol, 0.5% bromophenol blue, and 50 mM EDTA (pH 8)]. The reaction products were analysed by electrophoresis on a 1% agarose gel using a TBE buffer (89 mM Tris–HCl, 89 mM boric acid and 62 mM EDTA) at 1 V/cm, stained by 1 µg/ml ethidum bromide and photographed using a short wavelength UV lamp (ChemiImagerTM 5500 equipment, Alpha Inotech Corporation, CA, USA).
Densitometric analysis of the results were performed using the AlphaEasFC image processing and analysis software, and the percentage of topo I activity was calculated using the following equation: [1 – (sample/control)] x 100] as previously described (Bendetz-Nezer et al., 2004).
Topo II assay
DNA relaxation assay: increasing (or, alternatively, specific) concentrations of nuclear or cytoplasmic proteins were added to a topo II specific reaction mixture containing, at a final volume of 25 µl: 50 mM Tris–HCl, pH 8, 120 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 30 µg/ml BSA, 1 mM ATP, 10 mM EDTA and 250 ng pUC-19 DNA plasmid. Following incubation at 37°C for 30 min, the reaction products were analysed by 1% agarose gel electrophoresis as described earlier.
Decatenation assay: the assay is based on decatenation of kinetoplast DNA (kDNA) and since this assay is specific for topo II enzyme, it can be carried out with crude cell extracts. kDNA is a large network of plasmids, originated from trypanosome Crithidia fasciculata, therefore, when it is analyzed by gel electrophoresis, it penetrates only slightly into the agarose gel. Upon decatenation by topo II, mini circles monomers of DNA are formed.
Nuclear proteins were added to a specific topo II reaction mixture containing at a final volume of 25 µl: 50 mM Tris–HCl, pH 8, 120 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.5 mM EDTA, 25 µg/ml BSA, 1 mM ATP and 200 ng kDNA (TopoGEN, Port Orange, FL, USA). The reaction products were analyzed as described for the topo II DNA relaxation assay.
Determination of the size (MW) of topo I/topo II proteins by Western blot analysis
Antibodies: goat anti-human topo I polyclonal antibody (Bendetz-Nezer et al., 2004) was purchased from Santa Cruz (lot no. D101), Biotechnology Inc., CA, USA, mouse anti--actin antibody (Bendetz-Nezer et al., 2004) was from ICN Biomedical Inc., Irvine, CA, USA, (lot no. 8739F), and rabbit anti-human topo II antibody was from TopoGEN Inc. (Port Orange, FL, USA). Secondary antibodies were anti-goat IgG peroxidase (Santa Cruz, CA, USA), anti-mouse IgG peroxidase and anti-rabbit IgG peroxidase (ICN Biomedical Inc.).
Nuclear proteins derived from human sperm were analyzed by Western blotting as previously described (Sambrook et al., 1989; Kaufmann and Svingen, 1999) using either anti-topo I antibody (1: 2000), anti--actin antibody (1:1000) or anti-topo II antibody (1:2500). The immunocomplexes were detected by enhanced chemiluminescence (ECL) (Santa Cruz Biotechnology Inc., CA, USA). Densitometric analysis was performed as previously described. The level of topo I or topo II protein was calculated using the following equation: [topo I or II/ actin] x 100.
Two-dimensional Gel electrophoresis analysis
Two-dimensional gel analysis (2D-gel) was performed as described by O'Farell (O'Farrell and Goodman, 1976) using the Mini Protean 2D Electrophoresis cells and performed according to the manufacturer's (Bio-Rad Laboratories) instructions. Nuclear proteins (200 µg) were immunoprecipitated by anti-topo I antibodies and the immunocomplexes were subjected to 2D-gel analysis. Western blot analysis was performed with anti-topo I or anti--actin antibodies. The protein spots were detected by ECL (Santa Cruz Biotechnology Inc.).
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Topo I and topo II activity in isolated human sperm cells
To obtain a clear sperm sample, a Hypaque-Ficoll gradient was used to separate the sperm cells from debris, leucocytes and other accompanying cells (Ferrante and Thong, 1982
). The purity of the sample from leucocytes was verified by peroxidase assay, which is a common method for detection of leucocytes in the sample. No peroxidase activity was detected in the purified sperm samples. Topo activity of nuclear extracts was measured by the conversion of supercoiled plasmid to its partially relaxed or fully relaxed forms, as shown by representative pictures in Fig. 1. A typical ladder of DNA molecules (topoisomers), which are the products of topo I DNA relaxation activity, was observed in the nuclear extract derived from sperm cells (Fig. 1A, lanes 2 and 3). The sperm topo I activity demonstrates a nuclear protein concentration dependence since the DNA relaxation activity increased when a higher amount of sperm nuclear proteins were added to the reaction (compare lane 2 with lane 3). The presence of an ATP-dependent DNA relaxation activity, which is typical for topo II, was also demonstrated. Sperm nuclear extract was added to a specific topo II reaction mixture containing ATP. The results depicted in Fig. 1B (lane 3) show an ATP-dependent DNA relaxation activity which was lower than that observed with purified somatic human topo II (Fig. 1B, lane 2). Densitometric analysis of the results obtained from five different samples and experiments revealed that 95% (for topo I) and 80% (for topo II) of the supercoiled DNA molecules were converted to the ladder of partially relaxed molecules when 4 µg and 6 µg (respectively) of sperm nuclear proteins were used (Fig. 1C). At first, samples from different donors were separately analyzed for topo activity. Since no significant differences in the level of topo I or topo II activity between the various examined donors were observed (data not shown), the experiments were performed on a pool of sperm cells collected from at least three healthy and fertile donors.
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Characterization of the human sperm topo I
Topo I activity in human sperm cells was characterized by the following parameters.
Sensitivity to topo I inhibitors
Topo I activity was examined in the presence of 60 µM CPT, a known specific inhibitor of topo I (Pommier, 1998; Pommier et al., 1999; Li and Liu, 2000) and in the presence of DMSO, the solvent of CPT. The results depicted in Fig. 2A show that the conversion of the supercoiled plasmid to the relaxed form, decreased in the presence of CPT as evident by the intensity of the supercoiled band remaining after the reaction (compare lane 4 with lane 2). The addition of DMSO to the topo I reaction mixture only slightly affected the DNA relaxation of the enzyme (compare lane 3 with lane 2). These results show that the sperm DNA relaxation activity is inhibited by CPT which is a specific inhibitor of topo I and does not act on other DNA-binding proteins (Pommier, 2006).
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Size of the topo I protein
Nuclear protein extracts were prepared from sperm cells and from EBV transfected human lymphocyte cell line. Equivalent concentrations of nuclear proteins were analyzed by PAGE and by Western blot, using specific anti-human topo I antibody. The results depicted in Fig. 2B show that anti-topo I antibody reacted with an 85 kDa protein present in the sperm nuclear extract and with the 100 kDa topo I protein present in the lymphocyte nuclear extract. To examine the specificity of the antibody, the membrane was re-blotted, after a stripping procedure, with a solution containing anti-topo I antibody and topo I blocking peptide (which was used for the production of the anti-topo I antibody). The results depicted in Fig. 2C show that the topo I peptide blocked the recognition of the 85 kDa and the 100 kDa proteins present in the sperm and the lymphocyte nuclear extracts, respectively. These results suggest that the 85 kDa sperm protein is indeed recognized by the anti-topo I antibody, suggesting that sperm topo I is smaller in its molecular weight compared with the 100 kDa of the somatic topo IB enzyme protein. Further characterization of the sperm topo I protein was performed by 2D PAGE analysis. Nuclear sperm extracts were analyzed by the 2D PAGE following by Western blotting using anti-topo I antibody. As a control for our assay conditions, we examined the size and the basic/acidic characteristics of
-actin using specific anti--actin antibodies. The results depicted in Fig. 2D show that a spot of the size of 85 kDa in the basic region of the gel (pI 8.4–8.6) was observed when anti-topo I antibodies were used. Under the same assay conditions, as expected, one spot at the size of 40 kDa and in the acidic region of the gel was observed when anti--actin antibodies were used (Fig. 2E). The results suggest that sperm topo I is an 85 kDa basic protein as expected for a DNA binding enzyme.
Characterization of human sperm topo II
The presence of topo II protein in sperm has been previously reported (St Pierre et al., 2002). However, the examination of topo II activity and the characterization of this enzyme in the sperm were not investigated. Topo II activity, in human sperm cells, was characterized by the following parameters.
Sensitivity to inhibitors
Etoposides (VP-16) are known inhibitors of topo II which are used in cancer chemotherapy (Li and Liu, 2000). The sperm topo II DNA relaxation activity (Fig. 3A) as well as the somatic activity (Fig. 3B) were inhibited by 60 µM VP-16 which was added to the reaction mixture (compare lanes 3 with lane 2, respectively). No differences between the somatic and the sperm topo II activities in this respect was observed.
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Decatenation activity
Topo II is required for chromosomal segregation during cell division as separates between the chromosomes by its decatenation activity, and the catenation activity is needed for nucleosomal assembly (Wang, 2002
). The ability of the sperm topo II to decatenate kinetoplast DNA (a network of plasmid DNA) was examined. Decatenation activity is defined by the releasing of monomers (minicircles DNA) from the kDNA networks. Nuclear proteins extract derived from the examined somatic cells contained topo II which converted a kinetoplast DNA to monomer DNA molecules (Fig. 4A, compare lane 2 with lane 1). Under the same assay conditions, no decatenation activity was observed in nuclear extracts derived from human sperm (Fig. 4A, compare lane 3 with lane 2). The DNA band seen in the sperm sample, as well as in the somatic sample, is derived from the nuclear extract that contained residues of DNA fragments. The same band was observed when sperm nuclear extract was added to the reaction mixture depleted of kDNA (Fig. 4B, lane 3).
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The absence of sperm topo II decatenation activity might be due to the existence of decatenation inhibitor in the extracts derived from human sperm. Therefore, sperm nuclear proteins extract were added to a specific decatenation reaction which contained a purified somatic human topo II. The results depicted in Fig. 4B show that the addition of sperm nuclear extract inhibited the decatenation activity of the purified topo II (compare lane 2 with lane 1). This inhibition was specific to the sperm nuclear extract since sperm cytoplasmic extract did not affect the decatenation activity of purified topo II (data not shown).
Size of sperm topo II protein
Nuclear proteins derived from sperm cells were analyzed by Western blotting using a specific anti-topo II antibody. A single band of 97 kDa was observed in nuclear sperm extracts (Fig. 5A, lane 1). This protein is significantly shorter than the 170 kDa somatic topo II (Fig. 5A, lane 2). To examine the specificity of the antibody, competition-based experiments were performed. The anti-topo II antibodies were reacted with purified human somatic topo II protein prior to the Western blot analysis. A significant reduction in the band intensity of both the 97 kDa (in sperm extract) and the 170 kDa (in somatic extract) proteins (Fig. 5B compare with A) was observed. These results indicate that indeed the 97 kDa protein in sperm is recognized by anti-topo II antibody.
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DNA topoisomerases have been purified from several types of somatic cells, both normal and tumorigenic. The activities of these enzymes are predominantly needed for the various transactions of DNA including replication, transcription (Wang, 2002
; Leppard and Champoux, 2005). In non-dividing fully differentiated cells, such as neurons, a significant topo I activity and topo I protein is found (Plaschkes et al., 2005). Although sperm cells are non-dividing fully differentiated cells, they differ from neurons by the absence of transcription and translation processes. Therefore, the presence of topoisomerase activities in sperm is not trivial. The presence of topo II proteins in spermatozoa derived from mice were previously reported (Bizzaro et al., 2000; St Pierre et al., 2002; Shaman et al., 2006; Yamauchi et al., 2007). However, the topo activities and the properties of these enzymes in mature sperm cells, especially in human sperm, has not been investigated previously. The present results show that both topo I and topo II activities are present in nuclear extracts derived from purified human sperm. Characterization of the sperm topo I revealed that in vitro the enzyme relaxed negative supercoiled DNA and was inhibited by the somatic topo I inhibitor, CPT, suggesting that in these properties the sperm topo I resembles the somatic enzyme. However, the amount of sperm nuclear proteins required for the detection of this activity is 10-fold higher compared with that from somatic cells (Bendetz-Nezer et al., 2004). These results suggest that the level of topo I protein or its activity in the nucleus of human sperm is relatively low compared with that found in somatic cells. To determine the molecular weight and properties of the sperm enzyme, we used two gel electrophoresis methods: the SDS–PAGE and the 2D PAGE following by Western blotting with specific anti-human topo I antibody. The results showed that the sperm topo I is shorter in its size (85 kDa) compared with the somatic topo IB (100 kDa), and is a basic protein with a pI of 8.4–8.6 resembling the pI (8.2–8.6) of somatic topo I (Tournier et al., 1992). It is not yet clear if the 85 kDa protein represents a degradation product or a shorter version of the somatic 100 kDa topo IB enzyme. Various sizes of somatic topo I proteins have been reported in different cells which exhibit a DNA relaxation activity and were defined as degradation products of the full length topo I (Fleury et al., 1998; Bronstein et al., 1999). Therefore, it is more likely that the 85 kDa sperm topo I that possesses the DNA relaxation activity is actually a degradation product of the full size enzyme. Since sperm cells do not synthesize proteins, it is reasonable to suggest that the sperm topo I was synthesized during the spermatogenesis stages and remains in the mature spermatozoon.
Two topo II isoforms ( and ) are known, but since the reaction conditions are identical for both of them, it is not possible to distinguish between them when nuclear extracts are used. Therefore, the topo II activity present in the sperm nuclear extract could represent either or isoforms. Topo II decatenation activity is required, following DNA replication, for the segregation of the chromosomes. The sperm topo II possesses DNA relaxation activity, but lacks the decatenation activity. Moreover, sperm nuclear extracts contain inhibitors, the nature of which is unknown, of the decatenation activity of somatic topo II. Since sperm are fully differentiated cells, which do not replicate their DNA, they do not need the decatenation activity of this enzyme. Therefore, one may suggest that to maintain the condensed structure of the sperm chromatin, the activities of these enzymes should be prevented by the presence of inhibitors or by containing an enzyme that lacks the decatenation activity. The sperm topo II is similar in some characteristics to the somatic one, such as its sensitivity to the inhibitors of somatic topo II, etoposide VP-16. However, when we examined the size of the topo II protein using anti-topo II antibodies, we found that the sperm topo II is significantly shorter than the somatic enzyme (97 kDa protein compared with the somatic 170 kDa topo II protein). It is possible that the 97 kDa sperm protein is a degradation product of the 170 kDa full length topo II protein. St Pierre et al. (2002) showed that in mature sperm derived from mice, an 89 kDa protein was recognized by anti-topo II antibodies, which might be a degradation product of the full length topo II. However, the activity of the 89 kDa topo II enzyme in the mouse sperm was not investigated or characterized. To the best of our knowledge, there are no reports on degradation products of somatic topo II which exhibit a DNA relaxation activity, and no documentation of a somatic topo II enzyme that lacks a decatenation activity. Therefore, it is more likely that sperm might possess topo II with unique properties. It is not yet clear, what is the biological function of these enzymes in mature sperm cells, since in these cells no transcription or DNA replication processes are present. It is possible that the activities of these enzymes in the mature sperm are inhibited to maintain the condense structure of the chromatin. However, it was recently shown that mice spermatozoa contain a nuclear matrix associated topo II, which together with an extracellular nuclease in the presence of high concentrations of Mn+2/Ca+2, cleave the sperm DNA in an apoptotic like manner (Shaman et al., 2006). This suggests that under altered physiological conditions (i.e. high doses of divalent cations), the sperm topo II is involved in the digestion of the DNA which may represent an impaired function of this enzyme. Indeed, it has been shown that in mice, when topo II was induced to cleave the sperm DNA prior to fertilization, the oocytes injected with these spermatozoa failed to develop. These topo II mediated DNA breaks in sperm caused the degradation of the paternal DNA and did not affect the maternal DNA (Yamauchi et al., 2007).
During spermatogenesis, double-strand DNA breaks (DSB) have been observed to have been produced in part by the activity of topo II. These DSB are probably required for the remodeling of the sperm chromatin and the replacement of histones by protamines during spermiogenesis (Zhao et al., 2004; Laberge and Boissonneault, 2005a,b). Moreover, the addition of topo II inhibitors (teniposide) to the fertilization medium actually leads to the presence of endogenous DNA breaks in decondensing sperm, which suggests that complete remodeling of sperm chromatin during fertilization in mice might require the activity of topo II (Bizzaro et al., 2000).
Here, we show that both types of mammalian topoisomerases (I and II) are present in human sperm, but it is not yet clear what the roles of these enzymes are. Furthermore, the possible involvement of these enzymes in the re-organization of the human sperm chromatin to nucleosomes rapidly after gamete fusion should be investigated.
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Submitted on January 10, 2007; resubmitted on May 13, 2007; accepted on May 18, 2007.
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