doi:10.1093/humrep/dem170
Human Reproduction Vol.22, No.8 pp. 2183–2189, 2007
DNA topoisomerases I and II in human mature sperm
cells: characterization and unique properties
I. Har-Vardi1, R. Mali2, M. Breietman2, Y. Sonin1, S. Albotiano1, E. Levitas1,
G. Potashnik1 and E. Priel2,3
1
IVF unit, Department of Obstetrics and Gynaecology, Soroka University Medical Center, Faculty of Health Sciences,
Ben-Gurion University, Beer-Sheva 84105, Israel; 2Shraga 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: þ972-8-6479537; Fax: þ972-8-6479579; E-mail: [email protected]
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 IIa 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.
Keywords: decatenation; DNA relaxation; human sperm; topoisomerase I; topoisomerase II
Introduction
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 30 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 a
# The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.
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(170 kDa protein) and b (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 a 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.
Materials and Methods
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 –100106 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
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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 mg/ml
aprotinin, 2 mg/ml leupeptin, 1 mg/ml pepstatin A, 2 mg/ml antipain,
100 mg/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
48C 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,
48C, 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 ml: 20 mM Tris –HCl (pH 8.1), 1 mM
dithiothreitol (DTT), 20 mM KCl, 10 mM MgCl2, 1 mM EDTA,
30 mg/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 378C for 30 min, the reaction was
terminated by adding 5 ml 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 mg/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 2 (sample/control)]100] as previously described (BendetzNezer 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 ml:
50 mM Tris–HCl, pH 8, 120 mM KCl, 10 mM MgCl2, 0.5 mM DTT,
30 mg/ml BSA, 1 mM ATP, 10 mM EDTA and 250 ng pUC-19 DNA
plasmid. Following incubation at 378C 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 ml: 50 mM Tris –HCl, pH 8,
120 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.5 mM EDTA, 25 mg/
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.
Topoisomerases in human sperm
Determination of the size (MW) of topo I/topo II
proteins by Western blot analysis
Antibodies: goat anti-human topo I polyclonal antibody (BendetzNezer et al., 2004) was purchased from Santa Cruz (lot no. D101),
Biotechnology Inc., CA, USA, mouse anti-b-actin antibody (BendetzNezer 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-b-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/b actin] 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 mg) 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-b-actin antibodies. The protein
spots were detected by ECL (Santa Cruz Biotechnology Inc.).
Results
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 mg and 6 mg
(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.
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 mM 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).
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 b-actin using specific anti-b-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-b-actin antibodies were used (Fig. 2E). The results
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Figure 1: DNA relaxation activity of topo I (A), topo II (B) and % of topo activity (C) Nuclear proteins from sperm cells (2 and 4 mg) were added
to a specific topo I reaction mixture (A) and 6 mg of sperm nuclear protein or four units of purified topo II enzyme were added to a specific topo II
reaction mixture (B). Densitometric analysis was performed for the results obtained with 4 mg or 6 mg (respectively) nuclear proteins, and the % of
topo activity was calculated (C). The results are means + SD of five different experiments. S and R are the supercoiled and the relaxed forms of
the pUC 19 DNA plasmid, respectively; NE, nuclear extract
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 mM 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.
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
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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).
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.
Topoisomerases in human sperm
Figure 2: Properties of sperm topo I
(A) Inhibition of sperm’s topo I activity by CPT a specific topo I inhibitor: Nuclear proteins (2 mg) from sperm cells were added to a specific topo I
reaction mixture in the absence (lane 2) or presence of 60 mM CPT (lane 4) or the vehicle-1% DMSO (lane 3). (Lane 1 pUC19) pUC19 DNA only.
The reaction products were analyzed as described in the legend of Fig. 1. S and R are the supercoiled and the relaxed forms of the pUC19 DNA
plasmid, respectively; SNE, sperm nuclear extract. (B) The size (MW) of sperm topo I protein. Nuclear proteins from sperm cells (80 mg) and
nuclear proteins from human lymphocytes (40 mg) were analyzed by Western blotting using specific anti-topo I antibodies. (C) The specificity
of the antibody was verified by incubation of the antibody with its blocking peptide prior to the probing of the membrane. (D and E) 2D gel electrophoresis of proteins derived from sperm nuclear extract following by Western blot analysis using anti-topo I antibody (D) or anti-b-actin antibody (E). The blots were developed by ECL assay. AB, antibody; Lym, lymphocytes
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 IIa (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
Figure 3: Inhibition of sperm topo II (A) or purified somatic topo II
(B) DNA relaxation activity by etoposide, VP-16, a specific topo II
inhibitor
topo II inhibitor 1. Nuclear proteins (6 mg) from sperm cells or four
units of purified human topo IIa enzyme were added to a specific
topo II reaction mixture in the presence or absence of 60 mM
VP-16. (Lane 1) Supercoiled pUC19 DNA only. The reaction products
were analyzed as described in the legend to Fig. 1. S and R are supercoiled and the relaxed forms of the pUC 19 DNA plasmid, respectively; SNE, sperm nuclear extract
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.
Figure 4: Decatenation activity of topo II in sperm cells
(A) Somatic nuclear proteins (4 mg) (lane 2) and sperm nuclear proteins (6 mg) (lane 3) were added to a topo II reaction mixture containing 200 ng kDNA as the substrate. Lane 1—kDNA only. (B)
Inhibition of purified human somatic topo II activity by sperm
nuclear extract. Purified topo II (four units) were added to topo II reaction mixture containing kDNA (lane 1). Sperm nuclear extract (4 mg)
were added to the topo II reaction mixture containing kDNA and four
units of purified human topo II enzyme (lane 2). Sperm nuclear extract
only was added to a topo II reaction mixture from which the kDNA
was omitted (lane 3). NE DNA, nuclear DNA; SNE, sperm nuclear
extract
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Har-Vardi et al.
Figure 5: The size (MW) of sperm topo II protein
Sperm nuclear proteins (380 mg) (A, B left columns) and 250 mg of
nuclear proteins from somatic cells (A, B right columns) were analyzed on PAGE and by Western blot using specific anti-topo II antibodies (A). The specificity of the antibody was determine by
pre-incubation of the topo II antibody solution with purified human
topo II protein, followed by immunoprecipitation of topo II/AB
immunocomplexes with protein A—sepharose beeds (0.1 g/ml).
The supernatant was used for Western blot analysis as the source for
antibodies (B). SNE, sperm nuclear extract; NE, nuclear extract;
AB, antibody
Discussion
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 antihuman 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).
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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 (a and b) 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 a or b 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 IIa protein). It is possible that the 97 kDa
sperm protein is a degradation product of the 170 kDa full
length topo IIa protein. St Pierre et al. (2002) showed that in
mature sperm derived from mice, an 89 kDa protein was recognized by anti-topo IIa 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 IIb, which together with an extracellular nuclease in
the presence of high concentrations of Mnþ2/Caþ2, 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
Topoisomerases in human sperm
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 IIb 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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