Molecular Human Reproduction, Vol. 6, No. 7, 575-581,
July 2000
© 2000 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Heat stress reduces poly(ADPR)polymerase expression in rat testis
1 Department of Organic & Biological Chemistry, University Federico II of Naples, Via Mezzocannone 16, 80134-Napoli, Italy, and 2 Gamete Signalling Laboratory, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK
Abstract
Poly(ADPR)polymerase (PARP) is a chromatin-associated enzyme with a presumptive role in DNA repair during replication and recovery from strand breaks caused by genotoxic agents. It catalyses the attachment and elongation of ADPribose polymers (pADPR) to a variety of acceptor proteins (including PARP itself, and histones) and is particularly active in the testis where its expression varies according to the stage of germ cell differentiation. PARP degradation is also one of the classic indicators of apoptosis. In this investigation we have examined the effects of heat stress on the adult rat testis with respect to the concentration and activity of PARP, the nature of the pADRP nuclear acceptor proteins, the length of ADPR polymers and the activity of the ADPR depolymerizing enzyme, poly(ADPR)glycohydrolase (PARG). Our results show a significant reduction in the concentration and activity of PARP 4 and 8 days after artificial cryptorchidism, but no significant changes were observed in PARG activity or in pADPR length. Unexpectedly, the apoptotic degradaion of PARP was not detected following heat stress. These results confirm that PARP gene expression is developmentally regulated during spermatogenesis and indicate that it is suppressed coincidentally with the loss of meiotic spermatocytes during artificial cryptorchidism.
cryptorchidism/DNA repair/heat stress/PARP/testis
Introduction
It is well known that a lower scrotal temperature is required for a normal spermatogenesis, and that, with the exception of testicond mammals, exposure of the testis to abdominal temperature causes extensive degeneration of the germ cells (Gambacorta and Sfondrini, 1979
). Failure of spermatogenesis is also a feature of clinical cases of naturally occurring cryptorchidism in man and animals, although Leydig cells seem relatively insensitive (Gambacorta and Sfondrini, 1979). The germ cells most affected by heat stress are primary spermatocytes and early round spermatids (Abe, 1987). Despite extensive histological and cytological characterization of the heat-stressed testis, the mechanisms of germ cell loss are poorly understood. It has been reported that there is an increased incidence of apoptotic cell death following experimental cryptorchidism (Yin et al., 1997), and it is known that an early wave of apoptosis is required to maintain a proper ratio between differentiating germ cells and Sertoli cells. This `physiological' apoptosis is thought to be caused by high expression of apoptosis-promoting proteins coincident with the entry of type B spermatogonia into meiosis (Rodriguez et al., 1997).
Many genes are also known to be developmentally expressed in germinal cells especially in primary spermatocytes (Eddy and O'Brien, 1998). For example, several histone variants are cell- and stage-specific and have a role in modulating the chromatin changes that mediate replacement of the histone-rich nucleoproteins of spermatocytes to the protamine-rich nucleoproteins of the spermatids (Meiestrich et al., 1981; Kim et al., 1987; Drabent et al., 1998). The expression of several DNA-repair related enzymes also varies; the spermatocytes contain high concentrations of DNA polymerase ß (Alcivar et al., 1992) and a specific form of DNA ligase III (derived by alternative splicing), that is not able to interact with the repair-related factor, XRCCI (Mackey et al., 1997). In addition, p53 (which also appears to have a role in DNA repair), is also expressed at elevated concentrations during meiosis in primary spermatocytes (Schwartz et al., 1993).
Many of the aforementioned proteins have been functionally related to poly(ADPR)polymerase (PARP) which is a component of the nuclear protein complexes active during DNA replication and repair (Cadelcott et al., 1996; Simbulan-Rosenthal et al., 1996; Masson et al., 1998).
The existence of several PARP enzymes has been recently reported (Smith et al., 1998; Ame et al., 1999). The type II PARP is structurally distinct from the classical 113 kDa protein, as it is lacking most of the DNA-binding and the auto-modification domains (molecular weight 62 kDa; Ame et al., 1999). The type III PARP is a larger protein (molecular weight 142 kDa) containing ankyrin repeats linked to a PARP catalytic domain that is believed to regulate telomeric function (Smith et al., 1998). However, it seems that both such types of PARP contribute only partially to the poly(ADPribosyl)ation reaction in higher eukaryotes.
PARP is a versatile enzyme (for reviews, see Ohei et al., 1997, D'Amours et al. 1999). It is activated by DNA strand breaks to catalyse the modification of several DNA-binding proteins (including itself) with ADPribose polymers (pADPR) of varying length thereby reducing the nuclear pool of ßNAD. The pADPR homopolymer may be as long as 200 residues, may contain several branching points up to a proportion of 3% and may be linked covalently or non-covalently to a variety of nuclear proteins that include histone H1 and H2B, high mobility group proteins, nuclear matrix proteins, DNA polymerases, topisomerases, and p53 (Ohei et al., 1997; Malanga et al., 1998a,b).
pADPR has a high turnover depending on the functional state of the cell. Its breakdown is catalysed by poly(ADPR)glycohydrolase (PARG) which cleaves the ribose-ribose bond by an exoglycosidic hydrolysis following an endoglycosidic incision of the polymer (Ohei et al., 1997). Large polymers are degraded to smaller polymers in a fast and progressive reaction; further degradation then proceeds in what has been described as a slowly distributive reaction mode (Ikejima and Gill, 1988; Braun et al., 1994).
A physiological role for poly(ADPribosyl)ation of nuclear proteins is still unclear but recent approaches using antisense RNA (Ding and Smulson, 1994) and PARP-null mice (Wang et al., 1995) suggest it is important for the maintenance of genomic integrity especially following genotoxic stresses. A positive correlation between life span and PARP activity has been observed in mammalian species (Burkle et al., 1994). PARP is also a target for the interleukin 1-ß converting enzyme (ICE)-like specific proteases that are activated during the onset of apoptosis (Kaufmann et al., 1993).
In previous work (Quesada et al., 1996), we reported that PARP activity varies between different classes of germ cells being highest in primary spermatocytes and lowest in round spermatids. By contrast, there was little variation in the activity of PARG. In this study, we investigated the effects of heat stress on the expression of PARP and PARG in adult rat testis including the sub-cellular nuclear distribution and length of pADPR. We observed a decrease in PARP as a consequence of an early down-regulation of gene transcription but, unexpectedly, there was no increase in the apoptosis-related proteolysis of PARP protein.
Materials and methods
Reagents
[14C]-NAD+ nicotinamide (U14C adenine dinucleotide ammonium salt) (250 mCi/mmol) and [adenylate-32P]-NAD+ (1000 Ci/mmol) were supplied by Amersham International, UK. DNase I (EC 3.1.21.1), proteinase K (E.C. 3.4.21.64) phenyl methyl sulphonyl fluoride (PMSF), leupeptin, chimostatin, pepstatin, spermine and spermidine were obtained from Sigma Chemical Co, Italy. and all other chemicals were of the highest quality commercially available.
Unilateral experimental cryptorchidism
Adult male Wistar rats (~60 days of age) were used throughout this study. Surgery was performed under anaesthesia in accordance with Home Office, UK regulations. One testis + epididymis was drawn into the body cavity through a midline abdominal incision and restrained therein by a suture to the abdominal wall. The contralateral testis remained in the scrotum as a control.
Isolation of nuclei and chromatin fractions
Rat testis nuclei were isolated by homogenization and differential centrifugation (Quesada et al., 1996). Proteases were irreversibly inhibited by 10 µg/ml of leupeptin and chimostatin, 5 µg/ml of pepstatin and 1 mmol/l PMSF. The nuclear matrix isolation procedure was essentially as previously described (Quesada et al., 1994), based on a three times repeated extraction with a high salt buffer (2 mol/l NaCl, 0.2 mmol/l MgCl2, 0.1 mmol/l PMSF in 10 mmol/l Tris–HCl pH 7.5), preceded by a digestion with 250 µg/ml DNase I, of isolated nuclei (2 mg/ml of DNA) resuspended in 0.25 mol/l sucrose containing 50 mmol/l Tris–HCl pH 7.5, 10 mmol/l MgCl2, 60 mmol/l NaCl, 1 mmol/l PMSF, 10 µg/ml chimostatin and leupeptin, 5 µg/ml pepstatin. The nuclear matrix was then resuspended in one third volume of low-salt buffer (0.2 mmol/l MgCl2, 0.1 mmol/l PMSF in 10 mmol/l Tris–HCl pH 7.5).
Poly(ADP-ribosyl)ation reaction
Intact nuclei were resuspended (50x106 nuclei/ml) in 10 mmol/lM Tris–HCl pH 8 containing 0.25 mol/l sucrose, 14 mmol/l ß-mercaptoethanol, 10 mmol/l MgCl2, 60 mmol/l NaCl, 1 mmol/l PMSF, 50 µg/ml DNase I (reaction buffer) and incubated with 200 µmol/l NAD+ and 1 µCi/25x106 nuclei of [32P]-NAD+ for 20 min at 20°C. The reaction was terminated by chilling on ice and immediate centrifugation at 1000 g for 15 min at 4°C, followed by several washes with the reaction buffer. The different chromatin fractions were prepared from incubated nuclei by the procedure described above. The amount of [32P]-pADPR incorporated into acceptor proteins was determined by liquid scintillation counting (Beckman LS8100 Spectrometer, USA) of the radioactivity associated to the 20% trichloracetic acid-insoluble material, collected on a HAWP (0.45 µm, Millipore SPA, Italy) filter.
Poly(ADPR)polymerase and poly(ADPR)glycohydrolase assay
In the standard PARP activity assay, the reaction mixture (final volume 250 µl) contained 100 mmol/l Tris–HCl pH 8, 14 mmol/l ß-mercaptoethanol, 10 mmol/l MgCl2, 1 mmol/l PMSF, 12 µg DNase I, 200 µmol/l [14C]-NAD+ (10.000 cpm/nmole) and, as enzyme source, an amount of nuclei corresponding to 50 µg of proteins. After 20 min incubation at room temperature, the reaction was stopped by the addition of ice-cold thricloroacetic acid 40% (v/v) and the radioactivity associated to the acid-insoluble material counted on a Beckman LS8100 liquid scintillation spectrometer. One mIU is defined as the amount of enzyme activity catalysing the incorporation per minute of 1 nmole of ADPribose into acid-insoluble material.
The PARG activity assay was performed in a standard reaction mixture containing 50 mmol/l potassium phosphate (pH 7.2), 10 mmol/l ß-mercaptoethanol, 2.5 µmol/l [14C]-pADPR synthesized as described previously (Malanga et al., 1998b), and purified by dihydroxyboronate (DHB)–Biorex chromatography and, as enzyme source, an aliquot of cells corresponding to 30 µg of proteins in a total volume of 50 µl. After 10 min incubation at 37°C, the reaction was stopped by addition of an equal volume of ice-cold 40% trichloracetic acid and the radioactivity present in the acid-soluble material determined on a Beckman LS8100 liquid scintillation spectrometer. One enzymatic mU was defined as that liberating 1 nmole of ADPribose per minute.
Analysis of PARP reaction products
[32P]-radiolabelled ADP-ribose polymers incorporated into the nuclear proteins were detached by incubation at 37°C overnight with 10 mmol/l Tris–NaOH pH 12, 1 mmol/l EDTA in the presence of 0.2 mg/ml proteinase K. Samples were extracted with CHCl3/isoamyl alcohol (24:1), dried, and dissolved in 50% urea, 25 mmol/l NaCl and 4 mmol/l EDTA, pH 7.5. Finally, samples were analysed by electrophoresis on a 20% polyacrylamide gel (Panzeter and Althaus, 1990).
Analysis of pADPR acceptor proteins
Nuclear proteins contained in the TCA-insoluble fraction of nuclei incubated with [32P]-NAD were analysed by 5–18% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) gels as described previously (Quesada et al., 1994). Each labelled sample was run in duplicate to be either silver stained or transferred onto a nitrocellulose membrane using an electroblotting apparatus (Bio-Rad, Italy) before autoradiography (HyperfilmTM; Amersham).
Western blotting experiments
Western blotting was performed as previously described (Quesada et al., 1996) with 80 µg protein being loaded onto each lane of an SDS–PAGE gel. The nitrocellulose sheets were treated for 50 min at 37°C with the blocking solution (50 mm/l Tris–HCl pH 8, 0.15 mol/l NaCl, 0.5% Tween) containing 5% non-fat milk, and subsequently incubated with either rabbit anti-PARP polyclonal antibody (H-250 Santa Cruz) (dilution 1:6000) or rabbit anti-DNA polymerase ß (generous gift of Professor HSuzuki) (dilution 1:2000) or rabbit anti-DNA ligase III (generous gift of Professor A.Tomkinson) (dilution 1:10 000) for 2 h at room temperature, in the same solution containing 0.5% non-fat milk. The nitrocellulose sheets were then washed with the same solution without non-fat milk and incubated with the secondary antibody (alkaline phosphatase conjugate; Sigma) diluted 1:5000, for 60 min at room temperature, washed, and the enzyme activity detected using the immuno-star chemiluminescent detection system (Bio-Rad). Densitometric scanning was performed by the use of a Calibrated Imaging Densitometer (Biorad GS-710).
Protein and DNA assays
Protein concentration was determined using the Bradford protein assay reagent (Pierce, USA) with bovine serum albumin as a standard. Total DNA was measured by the diphenylamine method using calf thymus DNA as a standard.
Results
It is well known that loss of differentiating germinal cells occurs in the testis following exposure to abdominal temperature (heat stress). Figure 1 shows a histological section of a typical rat testis 4 and 8 days after artificial cryptorchidism. The lumen of seminiferous tubules become empty of spermatids and differentiating germ cells, and this is followed by loss of spermatocytes, leaving only the spermatogonia at the periphery of the seminiferous epithelium. Several biochemical parameters also correlated to loss of germ cells. Total DNA and total protein (mg/g tissue) decreased to ~50% of controls after 4 days with a further small decline after 8 days (results not shown).
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Activity of PARP and PARG in heat-stressed rat testis
In parallel to protein and DNA values, PARP activity (mIU/g tissue) decreased by 40% after 4 days of heat stress. When expressed in terms of specific activity (mIU/mg protein) PARP concentrations declined to 60% of initial value after 4 days and to 45% after 8 days (Figure 2
). In contrast, PARG activity did not change significantly between 1 and 8 days of heat stress (Figure 2, insert).
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Analysis of the concentrations of PARP, DNA polymerase ß and DNA ligase III in heat-stressed testes
To investigate whether the decrease in PARP activity was related to changes in expression of PARP protein, Western blots of nuclear extracts were probed with a polyclonal antibody raised against the catalytic domain of PARP. Figure 3A
shows that the amount of immunoreactive PARP is progressively reduced in testes subjected to different times of heat stress. Densitometric scanning of the band showed that 4 and 8 days cryptorchidism reduced PARP values to 54 and 35% of the control respectively.
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Figure 3B and 3C
show the expression levels of two other developmentally-regulated genes, DNA polymerase ß and DNA ligase III, the expression of which is stimulated during meiosis. As judged by densitometric scanning, the concentrations of both of these proteins were close to controls in 2 days cryptorchid testes, but they decrease thereafter reaching values that were 25% (DNA polymerase ß) and 45% (DNA ligase III) at 8 days of cryptorchidism.
Sub-nuclear distribution of PARP in normal and 4-day heat stressed cryptorchid testes
The Western blot analysis was extended to the different nuclear fractions that we have reported previously to contain different forms of the ADPribosylating enzyme (Quesada, 1998). Figure 4A shows the relative distribution of PARP in normal testis nuclei and in their subfractions, i.e. DNase I sensitive chromatin (Snt DNase I), DNase I resistant/high salt extractable chromatin and nuclear matrix. Different amounts of PARP characterize these fractions. Most of the enzyme was recovered in the DNase I sensitive chromatin where an additional band at ~90 kDa is also evident. This has been identified as the proteolytic fragment of PARP produced by the action of ICE-like proteases activated during the onset of apoptosis (D'Amours et al., 1999).
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The amounts of PARP in all the nuclear subfractions from 4 day heat-stressed testes were significantly reduced (Figure 4B
). Particularly noticeable was the decline in the DNase I sensitive fraction, and the disappearance of the characteristic 90 kDa apoptotic fragment.
Analysis of pADPR and its protein acceptors in heat-stressed testis
Another characteristic feature of the poly(ADP-ribosyl)ation reaction is the heterogeneity in length of the ADPR polymers. To investigate if there were any effects of heat stress on pADPR length, whole testis nuclei were incubated with [32P]-NAD and [32P]-ADPR polymers isolated as described in Materials and methods. The electrophoretic analysis (Figure 5) revealed similar pADPR size distribution patterns among control and heat-stressed testes. However, a slight enrichment in shorter polymers can be observed in testes from cryptorchid rats; pADPR from liver nuclei, which were used as controls, showed no significant changes in polymer size and distribution.
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[32P]-pADPR acceptor proteins in normal and heat-stressed testes were analysed by autoradiography, following electrophoretic separation of proteins from nuclei incubated with [32P]-NAD. Samples from the liver of the same animals were also analysed as a control. The results shown in Figure 6
showed that in testis the main pADPR acceptor is a protein that on the basis of its molecular weight could be PARP itself (molecular weight 113 kDa + ADPribose units). Moreover, after 4 and 8 days of cryptorchidism there was an increase in labelling of proteins with a molecular weight of 40–20 kDa, indicating a switch from auto- to hetero-modification reaction. These proteins were present also in liver both from normal and cryptorchid rats, and probably represent ADP-ribosylated histones.
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Discussion
This study has shown that disruption of spermatogenesis caused by heat stress in the unilaterally cryptorchid adult rat testis is accompanied by a decline in the activity of PARP but not of its counterpart PARG. Unexpectedly, the 90 kDa apoptotic fragment of PARP was not detected although cryptorchidism is known to induce apoptosis in germinal cells (Yin et al., 1997).
We cannot exclude the possibility that also the other PARP-like enzymes recently identified, PARP II (Ame et al., 1999) and tankyrase (Smith et al., 1998), are also affected in their activity by heat stress in rat testis. However, the immunoblot analyses agree with the enzyme activity assays suggesting that the 113 kDa classical PARP is the enzyme that is mainly regulated in cryptorchid testes.
A major aim of this investigation was to determine the molecular effects of heat stress on nuclear metabolism in germ cells by measuring the concentration of PARP. This protein, as well as DNA ligase III and DNA polymerase ß, is known to be differentially expressed during the development of male germinal cells (Alcivar et al., 1992). PARP is present at a high level in primary spermatocytes (Quesada et al., 1996), one of the principal cell-types in the testis, known to be very sensitive to elevated temperatures. In addition, PARP activity is stimulated by DNA strand breaks and its breakdown fragment at 90 kDa is one of the early markers of apoptosis (Kaufmann et al., 1993). Spontaneous apoptosis is particularly prevalent in the testes of prepubertal animals as a means of regulating germ cell/somatic cell ratios, but declines to a low concentration with maturity. In the adult mouse testis, DNA fragmentation was observed 6 days after unilateral cryptorchidism that preceded by 24 h the onset of germ cell loss and appearance of multinucleated giant cells in the seminiferous tubule lumen (Yin et al., 1997).
We have observed the presence of a 90 kDa apoptotic fragment of PARP in the DNase I sensitive chromatin of normal adult rat testis, probably representing the basal value of apoptosis observed by others. The apoptotic-specific proteolytic fragment of PARP normally represents only a small proportion of the total amount of enzyme present in whole nuclei and it becomes evident in the DNase I sensitive chromatin fraction where a higher amount of PARP is present (De Lucia et al., 1996). Its disappearance after 4 days of cryptorchidism, however, suggests that heat stress does not affect PARP breakdown, but rather its expression either at the transcriptional or translational level.
It is known that heat-induced stress results in the degradation of many mRNAs. A recent report (Mezquita et al., 1998) analyses the response of germ cells exposed to heat shock at the expression level, for Hsp70 and ubiquitin transcripts. The authors show that, in contrast to the lack of response of mammalian testis, the induction of the expression and polyadenylation of mRNA coding for such proteins, contributes to the development of thermotolerance during avian spermatogenesis.
Interestingly, a differential effect of heat stress on translation of mRNA for the histone H1t variant has been also reported (Cataldo et al., 1997) and the transition proteins TP1 and TP2 in rat primary spermatocytes and elongated spermatids respectively. The weaker resistance to abdominal temperature of the primary spermatocytes has been explained with a reduction in the proportion and in the size of polysomes translating H1t mRNAs. This might be a general mechanism since a reduction in the amount of nuclear components could stop the ongoing of differentiation in heat-stressed germ cells.
In previous studies, we have reported that the poly (ADPribosyl)ation reaction is functionally related to the ongoing of spermatogenesis in the rat (Quesada, 1998). In purified rat germinal cells we observed the maximum amount of PARP activity and amount in primary spermatocytes undergoing the pachytene phase of the meiotic division. Since these are also the cells most sensitive to the heat stress, the reduction of PARP observed in heat-stressed testis may be related to their specific loss. On the other hand, primary spermatocytes showed (Quesada et al., 1996) the same PARG activity as secondary spermatocytes and the round spermatids, yet the loss of such cells in heat-stressed testes does not alter the PARG activity values.
The use of the cryptorchid testis, containing mostly pre-meiotic undifferentiated germinal cells, allowed us to define that PARP enzyme has the same mode of action during all the stages of germ cell differentiation. Indeed, only slight differences were observed, after 4–8 days heat stress, both in the patterns of pADPR and its main acceptors. These slight differences were an increment of polymers not longer than 20 residues of ADPribose and to a higher proportion of hetero-ADPribosylation. The pADPR length may not be affected because the PARP enzyme still active in cryptorchid testis have a normal mode of action that is still able to catalyse a multistep reaction that encompasses the covalent attachment of one ADP-ribose unit to the g-COOH group of a glutamic residue of an acceptor protein (initiation reaction), the formation of a 1’-2' ribose–ribose glycosidic bond (elongation reaction) and the 1"-2’ ribose–ribose glycosidic bond (branching reaction) between ADP-ribose units (D'Amours et al., 1999). These findings can be related to the general notion that the auto-modification reaction occurs via long and branched polymers, whereas the hetero-modification is made by polymers not longer than 20 residues (Ohei et al., 1997).
PARP automodification seems then to be mainly related to an active germ cell differentiation. Such a result can be considered in the view that PARP is one of the components of the multi-protein complexes that represent the nuclear enzymatic machinery (Dantzer et al., 1998). Interestingly, we observed the co-regulation of PARP and two other nuclear enzymes (DNA polymerase ß and DNA ligase III) that are well-known partners of PARP. As already stated, the coincident over-expression of these and other proteins, e.g. XRCC-I and p53, functionally associated with PARP characterizes primary spermatocytes (Eddy and O'Brien, 1998). Thus, the over-expression of PARP and its partner enzymes might be related to the meiotic division and to the control of the homologous recombination that occurs during meiosis. A possible participation of the ADPribosylating enzyme in the recombination events and in the regulation of sister chromatid exchanges has been suggested for mammalian cells (Ohei et al., 1997).
In the light of such observations, it will be of interest to investigate whether the organization of the enzymes as components of multi-protein complexes, needed during the main nuclear events, is regulated by the PARP capacity to interact with them. It has been reported that it is the auto-modified form of PARP that contributes to dynamic recruitment of the nuclear machinery on specific chromatin sites (Althaus et al., 1999).
PARP has been considered to be important for genomic stability, dispensable in apoptosis (Wang et al., 1995), but essential for the survival of mice under genotoxic stress (Menissier-de Murcia et al., 1997). Moreover, the identification of a novel poly(ADP-ribosyl)ating enzyme (Ame et al., 1999) suggests a new level of complexity in the regulation of poly(ADPribosyl)ation system. The ability of the PARP form localized at telomers to function as a negative regulator of telomerase activity, suggests that the telomers role as biological clock in cells is regulated by poly(ADPribosyl)ation (Smith et al., 1998). Therefore, the PARP role as a guardian of genome integrity defined in somatic cells is even more important during spermatogenesis, when an aberrant DNA recombination could lead to the production of chromosomally-altered gametes. Our present results further contribute to the knowledge of the fundamental role of PARP on the control of the spermatogenesis in the rat.
Acknowledgments
The authors acknowledge Dr Alan Tomkinson and Dr Hisanori Suzuki for their generous gift of the anti-DNA ligase III and anti-DNA polymerase ß antibodies.
Notes
3 To whom correspondence should be addressed at: Dipt. Chimica Organica e Biologica, Via Mezzocannone 16, 80134-Napoli, Italy. E-mail: quesada{at}unina.it 
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Submitted on November 5, 1999; accepted on April 13, 2000.
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