Mol. Hum. Reprod. Advance Access originally published online on August 31, 2007
Molecular Human Reproduction 2007 13(11):821-828; doi:10.1093/molehr/gam062
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Differential contribution of poly(ADP-ribose)polymerase-1 and -2 (PARP-1 and -2) to the poly(ADP-ribosyl)ation reaction in rat primary spermatocytes
Department of Structural and Functional Biology, University ‘Federico II’, Via Cinthia Monte S. Angelo, 80126 Naples, Italy
1 Correspondence address. Tel: +39-81-679165; Fax: +39-81-679233; E-mail: quesada{at}unina.it
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Abstract |
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Poly(ADP-ribose)polymerases (PARP-1 and -2) are activated by DNA strand breaks to synthesize protein-bound ADP-ribose polymers from NAD+. The two enzymes are overexpressed in rat spermatocytes and are likely to play a role in meiosis. Indeed parp-2–/– mice, but not parp-1 knockouts, show hypofertility. Aside, PARP-1 and PARP-2 are both involved in DNA damage repair and signalling, but their relative contributions to such processes remain as yet unknown, largely because of the lack of PARP isoform-specific inhibitors that has precluded in vivo studies. Here, we used permeabilized rat primary spermatocytes or isolated spermatocyte nuclei and radiolabelled NAD+ to investigate potential isoform-specific effects on basic features of the poly(ADP-ribosyl)ation reaction, including size of ADP-ribose polymers at different NAD+ concentrations, extent of auto- versus etheromodification, and modulation of such reactions by the PARP inhibitor, PJ34. We found that PARP-1 automodification prevailed over PARP-2 modification. In addition, over 50% of cellular poly(ADP-ribose) was covalently bound to histones H1 and H2. The inhibitory effect of PJ34 appeared to be targeted mainly to the elongation step of the reaction. We propose that a different propensity of PARP-1 and PARP-2 to undergo automodification and/or catalyze etheromodification, both in terms of number of enzyme molecules being involved and amount of bound poly(ADP-ribose), may underlie distinct roles in the regulation of spermatocyte functions.
Key words: PARP-1/PARP-2/histone H1/histone H2/rat primary spermatocytes
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Introduction |
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Poly(ADPR)polymerases (PARP-1 and -2) are key players in processes that sense and repair DNA damage. Indeed, such a role may be particularly relevant to germinal cells, since during spermatogenesis genetic damage in the form of unrepaired DNA lesions may be transmitted via sperm to the offspring.
Germinal cells are characterized by the unique events of meiosis and it is known that: (i) DNA replication occurs in spermatogonia and prior to meiosis in primary spermatocytes, (ii) spermatogonia, spermatocytes and early spermatids are active in DNA transcription, whereas late spermatids and spermatozoa are totally inactive, (iii) an interval of regulated DNA nicking and repair (recombinational repair) occurs in primary spermatocytes, (iv) late spermatids are still able to repair their DNA damage before the final nuclear condensation.
Premeiotic spermatocytes undergo homologous recombination that begins with programmed double strand breaks (DSBs) at early prophase. The enzymatic DSBs response network described in mammalian germinal cells involves RAD 51, ATM, DNA-PK, histone H2AX, which are subjected to regulation by tumour suppressors like p53 (reviewed by Richardson et al., 2004).
Moreover, it is well established that the expression of DNA repair enzymes is developmentally regulated and most of them are overexpressed in premeiotic cells (Eddy and O’Brien, 1998). Previous reports showed that male germinal cells are characterized by an efficient base excision repair (BER): rat primary spermatocytes and round spermatids express similar levels of DNA glycosylases (Olsen et al., 2001) while DNA polymerase β and DNA ligase III are expressed at higher levels in primary spermatocytes (Di Meglio et al., 2003). Nucleotide excision repair (NER) activity in spermatogenic cell types is controversial (Jansen et al., 2001; Xu et al., 2005).
The participation of PARP-1 and PARP-2 in BER and NER pathways has been assessed in somatic cells (reviewed by Bürkle, 2005a). We have previously shown that PARP-1 and PARP-2 are expressed at high levels in rat primary spermatocytes (Atorino et al., 2000; Di Meglio et al., 2003); moreover, the two enzymes colocalize at the nuclear matrix (Tramontano et al., 2005).
Up to now, 18 members of the PARP superfamily (Amé et al., 2004; Burkle, 2005b; Hassa et al., 2006) have been identified; among them, PARP-2 (62 kDa) has been shown to co-operate with PARP-1 (113 kDa) for efficient BER (Schreiber et al., 2002) and in the signalling of DNA damage to survival/cell death pathways (Malanga and Althaus, 2005). PARPs mediate one of the immediate responses to DNA damage in eukaryotic cells by post-translational modification of nuclear proteins. In response to a genotoxic challenge, PARPs are activated following binding to DNA strand breaks and they catalyze the synthesis of poly(ADP-ribose) polymers (PAR) that may extend up to 200 residues in length. β-NAD+ is the substrate and PAR is covalently linked to chromosomal proteins, nuclear enzymes, transcription factors and mainly to PARP-1 itself.
The PARPs’ enzymatic counterpart catalyzing PAR catabolism is poly(ADPR)glycohydrolase (PARG). In rat germinal cells, two main forms of PARG have been reported: a full-length 110 kDa enzyme, i.e. mainly localized in the nucleus, and a 60 kDa isoform present in the cytosolic fraction (Di Meglio et al., 2003). However, under DNA damage conditions the latter rapidly accumulates in the nucleus to assure a rapid turnover of the polymer (Winstall et al., 1999). It is known that automodified PARP-1 is inactive; thus polymer degradation by PARG is required to restore PARP-1 catalytic activity.
The polyanion PAR can also compete with DNA for non-covalent interaction with nuclear proteins thereby modulating their functions (Pleschke et al., 2000; Malanga and Althaus, 2005). By such a mechanism, PARP-1 is able to recruit the DNA repair enzymatic machinery to DNA breaks or to silence transcription of damaged DNA by altering the function of transcription factors (TRFIIs) (Bürkle, 2005b). Moreover, by covalent poly(ADP-ribosyl)ation of the N- and C-terminal tails of histone H1 and H2B, PARP-1 and PARP-2 contribute to the relaxation of the 30 nm chromatin fibre, thus making breaks more accessible to repair complexes and/or transcription regulators (Bürkle, 2005a and references therein).
One of the most dramatic changes in chromatin structure is represented by the transition from nucleo-histone to nucleo-protamine organization of pre- to post-meiotic cells, i.e. preceded by expression of tissue- and phase-specific histone variants like H1t and TH2B (Eddy and O’Brien, 1998; van Roijen et al., 1998). We have found that in isolated rat spermatocyte populations, the tissue- and phase-specific H1t variant is the main poly(ADP-ribosyl)ation target (Atorino et al., 2000).
Several lines of evidence support a role for PARPs at specific stages of spermatogenesis. In fact, high levels of PAR have been detected in mouse spermatids at differentiation steps 11–14 in which the highest rates of chromatin nucleoprotein exchanges take place (Meyer-Ficca et al., 2005). Thereafter, PARP(s) expression reaches near undetectable levels in elongating spermatids (Aguilar-Mahecha et al., 2001) and ejaculated human spermatozoa (Taylor et al., 2004). It has been recently reported that genetic disruption of the PARP-2 gene in mice dramatically affects spermatogenesis; the loss of an essential role of PARP-2 during the prophase of the first meiotic division results in a massive apoptosis at the pachytene stage. PARP-2–/– spermatocytes also display a reduced number of cross-over, and the differentiation of spermatids is severely compromised (Dantzer et al., 2006).
Alteration in DNA damage signalling and repair is considered a common cause of infertility as well as of neoplastic transformation (Kobayashi et al., 2001; Agarwal and Allamaneni, 2005). Reactive oxygen species (ROS) are well known to initiate DNA strand breakage and have been closely associated with the aetiology of defective sperm function (Baker et al., 2003). By the use of PARP(s) and PARG inhibitors, we have already reported that PARP-1 activation following genotoxic stress (heat-shock, gamma-radiation, ROS) is required for the signalling and repair of DNA damage in rat primary spermatocytes in acute culture (Tramontano et al., 2000; Atorino et al., 2001; Di Meglio et al., 2004).
The present study was aimed at investigating the relative contribution of PARP-1 and PARP-2 to the poly(ADP-ribosyl)ation reaction in rat primary spermatocytes. Indeed such cells represent a useful system to study PARP-1 and PARP-2 mode(s) of action, however the lack of PARP isoform-specific inhibitors has not allowed in vivo analyses. We performed our analyses in permeabilized cells and isolated nuclei, after incubation with radiolabelled β-NAD+ given either alone (sub-micromolar concentration) or after mixing with unlabelled β-NAD+ to final concentrations ranging from 5 to 200 µM. The water-soluble quinazoline derivative, PJ34, was used as a general PARP inhibitor. We found that PARP-1 automodification prevails over PARP-2 modification. Moreover, over 50% of total poly(ADP-ribose) synthesized in permeabilized spermatocytes was covalently bound to histones H1 and H2.
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Materials and Methods |
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Reagents
-MEM culture medium from GIBCO Life Technologies was supplied by Invitrogen, Italy; streptomycin, penicillin and digitonin were from SIGMA-Aldrich, Italy; the cocktail of protease inhibitors was from ROCHE-Diagnostic, Italy; and PJ34 from Alexis Biochemicals was supplied by Vinci-Biochem, Italy.
Nicotinamide adenine [adenylate-32P] dinucleotide-[32P]-NAD+ (
1000 Ci/mmol, 10 mCi/ml) was supplied by Ge Healthcare, Italy.
Poly-vinylidene-fluoride (PVDF) membrane was from Millipore Corporation, USA.
Anti-PARP-1 rabbit polyclonal antibody (H-250), anti-DNA Topoisomerase II goat-polyclonal (N16), anti-Ku70 rabbit polyclonal antibody (H-30P), anti-XRCC-1 rabbit polyclonal antibody (H-300), anti-RAD-51 rabbit polyclonal antibody (H-92), anti-actin (I-19) and goat anti-rabbit IgG HRP-conjugate or goat anti-mouse IgG HRP-conjugate were purchased from SantaCruz Biotechnology Inc., UK. Anti-PARP-2 rabbit polyclonal from Alexis Biochemicals was supplied by Vinci-Biochem, Italy.
All other chemicals were of the highest quality commercially available.
Isolation and culture of rat primary spermatocytes
Testes were collected from 33 days old Wistar rats and populations of primary spermatocytes and round spermatids were purified by centrifugal elutriation as described by Quesada et al. (1996). Purity was >80% as assessed by FACS analysis for DNA content. After washing by centrifugation, cells were suspended in -MEM culture medium supplemented with 25 mM NaHCO3, 5% w/v fetal calf serum, 100 µg/ml streptomycin, 100 U/ml penicillin and plated at a density of 2 x 106 cells/ml into 4-well Nunc dishes. Cells were maintained in culture for 24–48 h at 32°C in 5% CO2/air and cell viability was assessed by Trypan Blue dye exclusion.
Nuclei isolation
Cells (5 x 106/ml) were suspended in a buffer containing 0.32 M Sucrose, 10 mM Tris–HCl pH 7.8, 2 mM MgCl2, 5 mM β-mercaptoethanol, 2 mM PMSF, 1% Triton X-100 and a 1:25 dilution of the protease inhibitors cocktail solution (Roche Diagnostics) prepared according to the manufacturer’s instructions. Cellular suspensions were homogenized in a Dounce homogenizer (five strokes) and centrifuged at 800g for 10 min at 4°C to separate nuclei (recovered in the pellet) from the cytoplasmic fraction (supernatant).
Poly(ADP-ribosyl)ation reaction
Spermatocytes were resuspended at 5 x 106 cells/ml in 50 mM HEPES buffer pH 7.5, containing 28 mM KCl, 28 mM NaCl, 2 mM MgCl2, 0.01% digitonin, 0.1 mM PMSF and a 1:25 dilution of the cocktail of protease inhibitors. Then, permeabilized cells were incubated at 30°C for 1 h with 5 µCi/ml [32P]-NAD+ (1000 Ci/mmol) alone or in the presence of unlabelled NAD+ 5, 50 and 200 µM, in the presence or absence of 10 µM PJ-34. The reaction was stopped by TCA addition (20% final concentration) and after 15 min standing on ice, cells were collected by centrifugation at 800g for 15 min, and washed twice with 5%TCA and three times with ethanol. Nuclei were subjected to the same treatment.
The cellular and nuclear pellets were resuspended in 40 mM Tris–HCl pH 7.8, 0.6 mM EDTA, 30 mM MgCl2, 0.1% Triton X-100, 1 mM β-mercaptoethanol, 10% Glycerol, 2 mM PMSF and a 1:25 dilution of the cocktail of protease inhibitors and stored at –20°C until use.
[32P]-PAR incorporated in the TCA insoluble fraction was counted in a Beckman LS8100 liquid scintillation spectrometer.
Protein concentration was determined using the Bradford protein reagent assay (BIO-RAD) with bovine serum albumin as a standard.
Poly(ADP-ribose) size analysis
[32P]-PAR was detached from protein by incubation at 60°C for 3 h in Tris–NaOH pH 12, extracted with CHCl3/isoamyl alcohol (24:1), dried in a SpedVac concentrator, dissolved in 50% urea, 25 mM NaCl, 4 mM EDTA pH 7.5 and finally subjected to 20% polyacrylamide gel electrophoresis and autoradiography.
Autoradiographic and immunological analyses
Aliquots of 120 µg of cellular/nuclear proteins were separated by SDS–PAGE (5–15% gradient gels) or acetic acid urea 15% PAGE (AU–PAGE).
Following separation by AU–PAGE, gels were equilibrated in 62.5 mM Tris-HCl pH 6.8, 5% β-mercaptoethanol, 3% SDS, 10% glycerol and subjected to a second dimension electrophoresis on 15% SDS–PAG. Proteins were either stained with Coomassie or transferred onto a PVDF membrane using an electroblotting apparatus (BIO-RAD).
The membrane was subjected to autoradiographic analysis by the PhosphorImager (BIO-RAD) and subsequently to immunodetection after blocking with 3% non-fat milk in TBST for 1 h. The following polyclonal antibodies were used: anti-PARP-1 (diluted 1:2000); anti-PARP-2 (diluted 1:5000); anti-DNA Topoisomerase II (diluted 1:200), anti-Ku70 (diluted 1:200); anti-XRCC-1 (diluted 1:1000); anti-RAD-51 (diluted 1:1000); and anti-actin (diluted 1:1000). All antibodies were diluted in 1% non-fat milk in TBST and incubations were for 2 h at room temperature. After washing 3x with TBST, membranes were incubated with peroxidase-conjugated mouse- or goat anti-rabbit IgG, diluted 1:4000 in 1% non-fat milk in TBST, for 1.5 h at room temperature. Finally, membranes were washed 3x in TBST and peroxidase activity was detected using the Luminol reagent (SantaCruz, UK). Images were acquired using the ChemiDoc (BIO-RAD) and band intensities were quantified by Quantity One.
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PAR synthesis in rat primary spermatocytes
Germinal cell populations were isolated from adult rat testis by centrifugal elutriation and their purity was assessed by FACS analysis, as previously described (Quesada et al., 1996). The identity of primary spermatocytes was further confirmed by analysis of the expression of the tissue- and stage-specific histone variant H1t (Atorino et al., 2000).
Permeabilized spermatocytes/isolated nuclei were incubated in the presence of [32P]-NAD either alone (sub-micromolar concentration) or mixed with different amounts of unlabelled NAD+ (final concentrations: 5–200 µM). The amount of [32P]-PAR incorporated in the TCA insoluble material was in all cases 0.5% of provided NAD+ and ranged from 5 to 200 picomol/µg of proteins (Fig. 1). The addition of PJ34 (10 µM) to the incubation mixture in the presence of 200 µM NAD+ reduced [32P]-PAR synthesis by 70% (data not shown).
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Furthermore, we determined the size of [32P]-PAR synthesized under such experimental conditions. Electrophoretic and autoradiographic analyses (Fig. 1B) of purified [32P]-PAR showed that at sub-micromolar concentration of [32P]-NAD+, (ADP-ribose)n ranged in size from monomers up to 8–10 ADP-ribose units in chain, whereas long and branched PAR molecules were synthesized at 5–200 µM NAD+. Identical [32P]-PAR size distribution patterns were observed in isolated nuclei under the same incubation conditions (data not shown).
PAR acceptors in rat primary spermatocytes
Poly(ADP-ribosyl)ated proteins in cellular lysates were identified by autoradiography following separation by SDS–PAGE and electroblotting onto a PVDF membrane. In addition, PARPs extensive automodification was determined by western blotting analysis as a reduction of the unmodified enzyme immunoreactive band. In fact, the increase in molecular weight due to multiple, long PAR molecules causes the protein to migrate slower and/or prevents its transfer onto PVDF membranes.
Autoradiographic analyses (Fig. 2A) showed that PARP-1 underwent efficient automodification at all tested NAD+ concentrations. However, the extent of automodification (number of PAR polymers/enzyme molecule and/or size of bound PAR) changed dramatically: in fact PARP-1 appeared as a 113 kDa radioactive band in the sample incubated with sub-micromolar concentration of [32P]-NAD+, but shifted to the top of the gel at a higher NAD+ concentration (200 µM). When incubation with 200 µM [32P]-NAD+ was carried on in the presence of 10 µM PJ34, we observed a full inhibition of the elongation reaction, but only a reduced effect on mono/oligo-ADP-ribosylation, as suggested by a restoration of the radioactive band at 113 kDa. Only a barely detectable signal was observed in the migration region of PARP-2 (62 kDa). The intensity of such a band was only slightly reduced after incubation with 200 µM NAD+ and almost unaffected by PJ34.
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Figure 2B shows the result of a western blotting analysis of the same samples. PARP-1 and PARP-2 were specifically detected by the respective antibodies and appeared as single bands in the region of the gel corresponding to their migration in the unmodified status. Band intensities were altered after incubation with 200 µM NAD+: by densitometric scanning, we determined a 75% reduction of the PARP-1 band following incubation with 200 µM NAD+, whereas only a 25% difference was found in the PARP-2 band intensities in the same samples. In both cases, the addition of PJ34 restored original PARP-1 and 2 band intensities.
Figure 2A also shows that a consistent fraction of [32P]-PAR in permeabilized spermatocytes is covalently bound to histone H1 and to a component of the core histones, most likely H2. Autoradiographic bands corresponding to such proteins were reduced after incubation with 200 µM NAD+, although a third band became evident, probably resulting from modification of histone H1 by longer PAR molecules.
To further substantiate the differences observed in PARP-1and 2 automodification levels as well as to confirm the identity of modified histones, the analysis was extended to spermatocyte nuclei, and proteins were separated by both 15% SDS–PAGE and 15% AU–PAGE. The latter electrophoretic system allows the separation of proteins by differences in their net positive charge at acidic pH and is more suitable for the analysis of basic proteins. Autoradiographic analysis following SDS–PAGE (Fig. 3A) showed that, although the types of acceptors were essentially the same as in permeabilized cells, the relative extent of their modification differed. At a sub-micromolar NAD+ concentration, automodified PARP-1 represented more than 70% of all ADP-ribosylated proteins. A smaller fraction of newly synthesized PAR was bound to PARP-2 and to a component of the histone core, and histone H1 modification was almost undetectable. Incubation with 200 µM NAD+ caused the disappearance of PARP-1 and PARP-2 bands, with simultaneous appearance of a signal at the top of the gel and a smearing of the core histone autoradiographic signal, possibly because of long and branched PAR molecules drastically altering acceptor protein masses.
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The same samples were analysed by AU–PAGE that allowed a better resolution of core histones, as shown by Coomassie staining of the gel (Fig. 3B). Autoradiography of electroblotted proteins did not show any radioactive signal in the sample from nuclei incubated with 200 µM NAD+; in fact, heavily poly(ADP-ribosylated) proteins are not expected to migrate in such an electrophoretic system. Conversely, oligo-ADPribosylation occurring at a sub-micromolar NAD+ concentration did not hamper protein migration and they appeared as radioactive bands/smear near the top of the gel, in correspondence with the core histones electrophoretic migration. By immunodetection (Fig. 3C), we could ascribe to PARP-1 and PARP-2 the radioactive signals near the top of the gel.
Furthermore, to get a more precise identification of the main PAR histone acceptors, we analysed the oligo-ADPribosylated sample by two-dimensional gel electrophoresis (Fig. 4B), with AU–PAGE and SDS–PAGE as first and second dimensions, respectively (Davies, 1982). Calf tymus histones were analysed in parallel (Fig. 4A) and taken as references for the identification of spermatocyte histones. Besides PARPs signals at the top of the gel, the autoradiographic analysis (Fig. 4B) showed a series of signals in correspondence with histone electrophoretic mobilities. Both H1b-e and H1a,t histone fractions were visible as well defined spots. Among the histone core components, histones H2B and H2A appeared to be modified; the smearing of the corresponding radioactive signals points to a heterogeneity in the ADP-ribosylation status of these proteins.
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The identification of labelled proteins as histones, initially based on their electrophoretic mobility in SDS- and AU–PAGs, was also confirmed by their solubility in diluted acids. In fact, the histone pool is extracted from rat primary spermatocytes by 0.2 M sulphuric acid as previously demonstrated (Atorino et al., 2000).
Levels and subcellular localization of potential PARPs targets/partners
In the light of the described functional association of PARPs with a number of nuclear proteins, we also looked at the endocellular levels of such proteins in rat primary spermatocytes and round spermatids. As shown in Fig. 5A, a number of proteins involved in the processing and/or signalling of DNA strand breaks, including XRCC-1, DNA-PK, RAD 51, p53, as well as PARP-1 and 2, DNA polymerase β and ligase III are expressed at higher levels in primary spermatocytes than in round spermatids. Conversely, we found that PCNA is more abundant in spermatids. DNA TOPO II, which was present at similar amounts in the two types of cell populations, was taken as a loading control. These observations were confirmed by densitometric scanning of the immunoreactive bands in several independent experiments (Fig. 5B).
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In this study, we further investigated some aspects of the poly(ADP-ribosylation) system in male germinal cells. We focused on rat primary spermatocytes, since it was already shown by us and by others, that PARP-1 and 2 enzymes are progressively lost in post-meiotic cells (Atorino et al., 2000; Di Meglio et al., 2003; Meyer-Ficca et al., 2005) and that the parp-1 gene is switched off in sperm (Aguilar-Mahecha et al., 2001; Taylor et al., 2004). These findings imply that PARP(s) enzymes exert their role of guardian(s) of genomic integrity at a crucial phase of germinal cell differentiation, such as the meiotic division. Indeed, at meiosis a network of DNA repair enzymes have a prominent role in mammalian spermatogenesis (Eddy and O’Brien, 1998) and epigenetic histone modifications also play a crucial role at meiotic and post-meiotic stages (Maleki and Keeney, 2004). Remarkably, poly(ADP-ribosyl)ation contributes substantially to both DNA repair mechanisms and epigenetic information (Burkle, 2005b). However, the relative contribution of PARP-1 and -2 enzymes is still elusive.
To gain insights on nuclear PARPs mode of action, we used purified populations of rat primary spermatocytes (Atorino et al., 2000). Preliminarily, we investigated poly(ADP-ribosyl)ation reaction features in our experimental conditions, and we found that even in vitro (permeabilized cells and nuclei), the reaction is strictly regulated both in terms of reaction products and protein acceptors. The synthesis of PAR of high size and complexity was stimulated by micromolar amounts of NAD+, whereas short oligomers of ADP-ribose represented the main product at sub-micromolar NAD+ concentrations. Furthermore, we found that in permeabilized cells, independently of substrate concentration, the main acceptors were PARP-1, histone H1 and a component of the histone core, most likely H2A/B, whereas only a minor fraction of PARP-2 underwent modification. Interestingly, PJ34, a widely used inhibitor of PARP activities, appeared to affect mostly the elongation step of the reaction.
Noteworthy, although the types of acceptor proteins were the same in permeabilized cells and isolated nuclei, the relative modification extent of these proteins differed. In particular, at a sub-micromolar NAD+ concentration, PARP-2 modification levels appeared to be higher in isolated nuclei than in permeabilized cells, whereas histone modification was reduced in the former. In any case, however, PARP-1 automodification prevailed over PARP-2 modification. Moreover, at a higher NAD+ concentration, elongation of PAR molecules occurred both on PARPs and histones to a higher degree in isolated nuclei than in permeabilized cells. Such differences may be explained as a consequence of subtle changes in chromatin structure occurring during nuclei isolation (Belikov and Wieslander, 1994).
As poly(ADP-ribosyl)ation of PARP(s) causes an electrophoretic shift towards the top of the gel and consequent depletion of the corresponding immunoreactive bands; by densitometric scanning quantification, we could estimate that three-fourth of PARP-1 molecules and one-fourth of PARP-2 molecules underwent extensive modification. It has been reported (Amé et al., 1999) that recombinant mPARP-2 has a 18-fold lower activity than PARP-1 and can support 25% of PAR synthesis, in the absence of PARP-1; then a backup role of PARP-2 in somatic cells was postulated. This does not seem to apply to germinal cells since PARP-1–/– mice are fertile, whereas PARP-2–/– mice suffer severe hypofertility suggesting that the physiological role of PARP-2 may extend beyond its known involvement in the response to DNA damage, and entails a function both in meiosis and in post-meiotic germinal cell differentiation (Dantzer et al., 2006). Recently, using RNA interference to down-regulate PARP-1 and/or PARP-2 in human cells, Fisher et al. (2007) demonstrated that PARP-2 is dispensable for cellular recovery from oxidative DNA damage. In this study, we have shown that PARP-1 is the primary source of PAR synthesis in isolated rat primary spermatocytes with both the enzyme itself and histone proteins serving as covalent PAR acceptors; only a minor portion of newly synthesized PAR was found to be covalently bound to PARP-2. In line with the data reported earlier, these results indicate potential distinctive roles of PARP-1 and PARP-2 in germinal cells.
We also investigated the ethero-poly(ADP-ribosyl)ation of histone proteins. Both H1 and H2 components appeared to be modified in primary spermatocytes. Indeed, histones are well known PAR acceptors among low molecular weight nuclear proteins, and their identification can be based on their well-established electrophoretic behaviour both in SDS- and AU–PAGEs (Davie 1982). Interestingly, the NAD+ dependent elongation step of poly(ADP-ribosyl)ation occurred also on histones, although to a lesser extent than on PARPs. Furthermore, the fact that the histone poly(ADP-ribosyl)ation signal was still detectable after 2D-PAGE and/or electroblotting onto PVDF membrane strongly support the covalent nature of the histone-PAR bond.
We have previously reported that in primary spermatocytes the testis-specific histone H1 variant, H1t may either undergo covalent mono-/oligo-ADPribosylation or engage in non-covalent interactions with long PAR molecules (Atorino et al., 2000; Malanga et al.,1998). In this report, we show that core histones are also PARP targets for covalent poly(ADP-ribosyl)ation both in permeabilized cells and isolated nuclei. The latter represented a useful system to confirm the identity of ADP-ribosylated low molecular weight proteins as histones H2A and H2B. It remains to be established whether PARP-1 and PARP-2, besides having different automodification capacities, also have histone-specific targeting activities in rat germinal cells.
Indeed, epigenetic regulation of chromatin structure by poly(ADP-ribosyl)ation can play a role during nucleo-histone to nucleo-protamine transition in meiotic spermatocytes. We already postulated that such a transition could be regulated at the chromatosomal level by modification of the phase-specific H1t histone variant, exclusively expressed in primary spermatocytes (Atorino et al., 2000). Moreover, even the nucleosome disassembly of core histone to allow for substitution by protamines would be facilitated by their poly(ADP-ribosyl)ation. Future analyses will be addressed at determining whether testis-specific variants of H2 histones are involved.
Another class of PARPs partners is represented by DNA repair/replication factors, which can be both covalently and non-covalently modified by PAR, and physically interact with PARP-1 and -2 (Schreiber et al., 2002). We found that primary spermatocytes have higher expression levels than round spermatids of XRCC1 as well as of DNA polymerase β and DNA ligase III, confirming the fundamental role of BER in such germinal cells; moreover, other proteins involved in the processing and/or signalling of DNA damage (i.e. DNA-PK, RAD51, p53) were also present at higher amounts in spermatocytes. Such phase-specific high expression of PARP partners could also contribute to the different enzymatic properties of PARP-1 and -2 in premeiotic spermatocytes.
In conclusion, while confirming the high ADP-ribosylation capacity of primary spermatocytes, our study provides new information regarding a likely different mode of action of PARP-1 and -2: in particular, results point at a different propensity of the two enzymes to undergo automodification, in terms of both number of enzyme molecules being involved and number/size of ADP-ribose polymers/enzyme molecule. It shall be interesting to determine whether the two enzymes also differentiate in their etheromodification target specificities. These differences could underlie distinctive roles of PARP-1 and PARP-2 in the regulation of meiotic cell functions.
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This work was supported by MURST-PRIN 2004–2006 funding.
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References |
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Submitted on July 25, 2007; accepted on August 21, 2007.
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