Intracellular distribution of poly(ADP-ribose) synthetase in rat spermatogenic cells

Experimental Cell Research 180 (1989) 353-366

Intracellular

Distribution of Poly(ADP-ribose) Rat Spermatogenic Cells

Synthetase

in

ILONA I. CONCHA,” JAIME FIGUEROA,‘” MARGARITA I. CONCHA,” KUNIHIRO UEDA,? and LUIS 0. BURZIO*” *Institute de Bioquimica, Facultad de Ciencias, Uniuersidad Austral de Chile, Valdivia, Chile, and TDepartment of Clinical Science, Kyoto University Faculty of Medicine, Kyoto, Japan

The highest activity of poly(ADP-ribose) synthetase was found in the testis among several rat tissues tested. Subcellular fractionation of the testis demonstrated that the synthetase was localized primarily in the nucleus and partially in the microsomal-ribosomal fraction. This result was confirmed by immunocytochemical staining with the enzymespecific antibody. The synthetase was localized in the nuclei of interstitial cells, Sertoli cells, spermatogonia, and spermatocytes. In addition, round spermatids showed a granular staining in the cytoplasm, which was comparable in intensity with that in the nucleus. The cytoplasmic synthetase had a molecular weight of 115,000 and synthesized oligomers of ADP-ribose on itself (automodification). The synthetase activity in the isolated cytoplasmic fraction was stimulated about threefold by the addition of DNA and depressed by treatment with DNase I, suggesting the presence of endogenous activator DNA. A candidate DNA for such an activator was isolated from the microsomal-ribosomal fraction, and identified tentatively as mitochondrial DNA on the basis of its size and restriction fragment patkITE.

0

1989 Academic

Press, Inc.

Poly(ADP-ribose) synthetase is an enzyme which catalyzes formation of a nucleic acid-like homopolymer, referred to as poly(ADP-ribose), using NAD as a substrate [ 11. The oligomers and polymers of ADP-ribose are covalently attached to either the enzyme itself [2-51 or various nuclear proteins including histones [6-lo]. Although the biological roles of the enzyme and its product have not been fully established, they have been implicated in several cellular events such as DNA repair [l l-131, cell differentiation [14-161, gene expression [17, 181, and chromatin structure [ 19-221. Spermatogenesis is a complex process of cell differentiation which involves proliferation and renewal of spermatogonia, chromosome pairing and recombination during meiosis, and generation of haploid spermatids during spermiogenesis [23]. During this process, a variety of biochemical events such as DNA replication, DNA repair, transcription, and extensive and unique changes in chromatin structure take place. These events might be coordinated and modulated by poly(ADP-ribosylation) of proteins. Hence, spermatogenesis offers an attractive model for studying the relationship between these cellular events and poly(ADPribose) metabolism. We examined the level of poly(ADP-ribose) synthetase activity in rat testis, and found that the specific activity of the enzyme (enzyme ’ To whom reprint requests should be addressed. 353

Copyright @ 1989 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827189 $03.00

354

Concha

et al.

unitsimg of protein) was much higher than in ail other major organs also found, by means of subcellular fractionation and immuno~yto~ cedures, the presence of a significant level of enzyme activity in t spermatogenic cells, especially in round spermatids. MATERIALS

AN

s

Materials. [adenosine-‘4C]NAD (sp act 534 mCiimmo1) was purchased from New England Nuclear. Sucrose (grade I), pancreatic DNase I and RNase, phenylmethylsulfonyl fluoride (PMSF), sodium dodecyl sulfate (SDS), goat anti-rabbit immunoglobulin G (IgG) (alone or conjugated with peroxidase), a peroxidase-anti-peroxidase (rabbit) complex, and other biocbemicals were from Sigma Chemical Co. Restriction enzymes, EcoRI and BarnHI, and k DNA-NindIII fragments were from Bethesda Research Laboratory. Nitrocellulose (BA 85) was obtained from Schleicher & Schull. Supplies for electron microscopy were from Polysciences, and other chemicals were from E. Merck (West Germany). Extraction ofpoly(ADP-ribose) synthetase. Male albino rats were sacrificed by cervical dislocation, and various organs were excised. After removal of the fat tissue, the organs were homogenized in 5 ~01 (V/W) of a medium containing 50 mJ4 Tiis-HCl, pH 7.5, 2 nnV dithiothreitol (DTT), 0.5 mM PMSF, and 100 mJ4 MgC&. This procedure was catied out at 4°C with a Dotnce homogenizer. The homogenate was centrifuged at 15,000g for 15 min at 4”C, and the postmitochondrial supernaiant was centrifuged for 120 min at 140,OOOg.This supernatant represents the crude extract of the enzyme. Subcellular fractionation of rat testis. The testis was decapsulated and homogenized in 10 vol of ice-cold medium containing 0.25 M sucrose, 50 mM Tris-HCl, pH 1.5, 50 mM KCl, 5 mM 0.5 mM PMSF. This procedure was carried out with a Potter-Elvehjem tissue grinder equipped with a Teflon pestle. The homogenate was centrifuged at 1OOOg for 15 min at 4°C to obtain the crude nuclear fraction, and the resulting supernatant was centrifuged at 9000g for 15 min at 4°C to obtain the mitochondrial fraction. The postmitochondrial supernatant was either centrifuged directly at ; lO,OOOg or applied on top of a 2 M sucrose cushion containing 50 m&f Tris-HCl, pH 7.5, 50 rnil/l KCl, and 5 mM MgCI,, and centrifuged at the same speed for 2 h at 4°C. The compact pellet obtained with the first procedure or the layer at the 2 M sucrose interface was referred to as the microsomal-ribosomai (M-R) fraction of the testis. Assay of poly(ADP-ribose) synthetase activity. The activity of poly(ADP-ribose) synthetase was determined as described [24], except that 100 pg of calf thynms DNA was added to the reaction mixture (50 ~1)in the case of crude extract. One enzyme unit is the amount of enzyme that catalyzes the incorporation of one micromole of [?Z]ADP-ribose per minute at 25°C. Polyacrylamide gel electrophoresis. Slab (150x150~ 1.5 mm) polyacrylamide gel electrophoresis in the presence of SDS was performed using the discontinuous buffer system described by Laemmii [25]. The gel was stained as described before [26]. The standards of molecular weight used were /3galactosidase (M, 116,000) phosphorylase b (k&92,500), bovine serum albIumin (&& SS,OOO),ovdbumin (.Mr 45,000), carbonic anhydrase (M, 29,000), and lysozyme (M, 14,300). The distribution of radioactivity in the gel was determined by slicing the gel strip into 2-mm pieces, dissolving each piece in 0.5 ml of 30% H202 [27], and counting the dissolved material in a T&on X100-based scintillation mixture [28]. Western blot analysis. For this and other procedures, an antibody specific against calf thymus poly(ADP-ribose) synthetase was prepared in rabbit 1291.The characteristics of this antibody ha.ve been described elsewhere [29, 301. No difference was noticed between the antiserum or the IgG fraction used in these studies. To immunodetect the enzyme in the M-R fraction, the proteins were separated by SDS-polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose according to the procedure of Tsang et al. [31]. The blotted nitrocellulose sheet was exposed for 4 h to anti-synthetase antibody (diluted 1: 10,000in saline-phosphate buffer, pH 7.0). The sheet was then washed and exposed to anti-rabbit IgG conjugated with peroxidase. The procedure was completed with the substrate color development

Pll. Immunocytochemistvy. Male albino rats (200 to 250 g body weight) were anesthetized with nembutal and perfused via the abdominal aorta with physiological saline (100 ml) followed by 200 ml of cold aqueous Bouin’s solution. The testes were excised and further fixed by immersion in the same fixative overnight with continuous agitation. The fixed tissue was dehydrated with ethanol, cleared with acetone, and embedded in methacrylate. Sections of I km thickness mounted on glass slides were

Poly(ADP-ribose) synthetase in spermatogenic cells

355

incubated with anti-synthetase antibody (diluted 1 : 1000)for 8 h at room temperature. The slides were washed three times with saline-phosphate buffer, and then incubated for 30 min with anti-rabbit IgG produced in goat (diluted 1 : 100). After incubation with the second antibody and washing, the samples were incubated for 30 min with the peroxidase-antiperoxidase complex (PAP, diluted 1: 75) according to the procedure described by Sternberger et al. 1321.Finally, the samples were incubated with diaminobenzidine and H202 [32] and viewed with a Zeiss photomicroscope. Isolation of DNA. The M-R fraction was resuspended in a solution containing 50 mit4 Tris-HCl, pH 7.5, and 10 mM EDTA, adjusted to 1% SDS, and incubated at 37°C for 3 h with 100 ygiml of proteinase K. The lysate was extracted once with one volume of phenol-chloroform-isoamyl alcohol (25 : 24: 1) at room temperature for 15 min. After centrifugation, the aqueous fraction was reextracted with one volume of chloroform-isoamyl alcohol (24 : 1). Half a volume of 7.5 M ammonium acetate was added to the final fraction, and nucleic acids were precipitated with 2 vol of ethanol at -20°C overnight. The nucleic acids recovered by centrifugation were dissolved in a small volume of a solution containing 10 mM Tris-HCl, pH 8.0, 1 n&f EDTA, and 0.15 M NaCl, and incubated with RNase (SOug/ml) at 37°C for 2 h. The sample was extracted with phenol, and DNA was precipitated with ethanol as described. The final pellet of DNA was resuspended in a solution of 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 0.15 M NaCl. Mitochondrial DNA (mtDNA) was puri’iied according to the procedure described by Van Tuyle and McPherson 1331. Digestion with restriction enzymes. The DNA purified from mitochondria or from the M-R fraction was digested with 10 units of EcoRI or BumHI for 2 h at 37°C. The reaction medium was prepared following the manufacturer’s recommendation. Analysis of the purified DNA and the digestion products was carried out by agarose gel electrophoresis in a horizontal apparatus. The concentration of agarose was 1%, and the electrophoretic buffer contained 40 mM Tris, 20 n&f acetic acid, and 2 mM EDTA, adjusted to pH 8.1 [34]. After electrophoresis for 7 h at 50 V, the gel was stained for 15 min with 0.5 ugiml of ethidium bromide in water, and photographed under ultraviolet light using Polaroid 667 Land pack film. 1 DNA digested with Hind111 was used as molecular weight standard. Other determinations. Protein was determined by the method of Lowry et al. [35] using bovine serum albumin as standard. The average chain length of oligomers of ADP-ribose was determined by a modification [6] of the procedure described by Shima et al. [36]. The amount of DNA per gram (wet weight) of tissue was determined as reported [37]. Electron microscopy. A pellet of the M-R fraction was fixed for 2 h in 2% glutaraldehyde buffered with 100 m&4 sodium-phosphate at pH 7.5. After a rinse with the same buffer, the samples were posttixed in 1% 0~0~ for 2 h, rinsed with buffer, dehydrated with ethanol and acetone, and embedded in Epon-Araldite. Thin sections were stained with uranyl acetate and lead citrate and examined in a Phillips EM-300 electron microscope.

RESULTS Distribution of Poly(ADP-ribose) Synthetase As shown in Table 1, of the rat organs studied, the testis had the highest specific activity of poly(ADP-ribose) synthetase. This result was similar to that of Agemori et al. [38], although they used mouse tissue and a different extraction procedure. Moreover, if the enzyme activity was expressed in relation to the amount of DNA in the homogenate, once again the testis showed the highest specific activity (Table 1). The extraction procedure reported here was quite efficient. With 100 mM MgC12, more than 90% of the enzyme activity present in the homogenate was recovered in the final crude extract (see Materials and Methods). A similar portion of enzyme activity was recovered if the tissue was extracted with 150 mM MgC12,while only 40 % was obtained with 50 mM. In contrast to other extracts so far reported, the enzyme activity in our MgCl? extract was almost completely dependent on DNA; the activity in crude extracts of testis, liver, brain, and lung assayed without addition of exogenous DNA was about 3 % of the activity

3.56 Concha et al.

Poly(ADP-ribose) synthetase activity in extracts of various rat organs Specific activity (ymol X IO’) Organ Testis Spleen Liver Lung Brain Kidney

(mg of proteinimin) -DNA +DNA 1.280 0.689 0.174 0.144 0.129 0.076

0.035 0.240 0.004 0.008 0.003 0.056

(mg of DNA/mm) +DNA 8.7!8 2.058 2.352 1.286 2.200 0.844

Mote. Homogenates were prepared in a medium containing 100 m&I MgClz, as described under Materials and Methods. Assay was carried out for 5 min at 25°C with or without calf thymus DNA (100 ngim!). Each value represents the average of two determinations, and they are expressed as micromoles of NAD incorporated per milligram of protein or per milligram of DNA.

assayed with DNA (Table 1). The extract fro dependency on DNA, which might be due to these extracts; homogenization of these tissues cult than others, Since germinal cells represent the most abta testis (cf. Fig. 2B), the high level of the synkhetase ac should be associated with the spermatogenic cells. It was also important to distribution of the enzyme. As shown in Table 2, §~bcell~lar ~ract~~~at~~~of the testis indicated that the number of enzyme units in t one-fifth that in the nuclear fraction. In contrast, the fraction was equal to or higher than that in a unique situation in the testis, since a n detected in the M-R fraction of liver, brain, and the specific activity in other fractions was mu Electron microscopy of the M-R fracti TABLE 2 Subcellular distribution of poly(ADP-ribose) syzthetase k the testis Fraction Homogenate Nuclear fraction Mitochondrial fraction M-R fraction Final supematant

Total activity (units X if?)

Specific activity (units X 103/mgof protein)

28.03 33.45 0.46 6.20 1.66

3.50 9.75 2.70 13.37 0.78

Note. Rat testis was homogenated and fractionated as indicated under Materials and Methods. Aliquots of each fraction were assayed for poly(ADP-ribose) synthetase activity without addition of DNA. The results are the average of two determinations obtained from two independent preparations.

Poly(ADP-ribose)

synthetase

in spermatogenic

cells

357

Fig. 1. Electron micrograph of the M-R fraction of rat testis. A pellet of the M-R fraction obtained by centrifugation at 110,OOOgwas fixed and processed as described under Materials and Methods. Abundant microsomal vesicles are shown at the upper part. The bottom shows masses of ribosomelike particles. Bar= 1 pm.

(Fig. 1). Only abundant vesicles and large masses of particles of about 20 nm were observed. These materials were not glycogen, since identical particles were observed in the M-R fraction prepared from testis of fasting rats. Hence, these particles correspond most probably to large aggregates of ribosomes. Immunocytochemical

Studies

The above results demonstrated the presence of poly(ADP-ribose) synthetase activity in the M-R fraction of testis. However, it is possible that the presence of the enzyme in the cytoplasm might be the consequence of leakage from the nucleus. To examine this possibility, the immunolocalization of the enzyme was carried out. Sections of testis or liver were incubated with an antibody specifically recognizing the synthetase, and the bound first antibody was visualized using the peroxidase-antiperoxidase technique [32]. In agreement with previous reports [30], rat liver nuclei were clearly stained (Fig. 2A). In the testicular section, the nuclei of interstitial cells, spermatogonia, Sertoli cells, and spermatocytes were well stained (Fig. 2B). In pachytene spermatocytes, the reaction was very intense, especially with condensed chromosomes (Fig. 2C). In contrast, the staining of the nuclei of round spermatids was less intense (Fig. 2C), and was negative in elongated spermatids and sperm (Fig. 2B). An important finding was the intense granular staining of the cytoplasm of some spermatogenic cells (Figs. 2B and C). This was particularly clear in round spermatids where the reaction formed a sort of capping of the nucleus facing the lumen of the seminiferous tubule (Fig. 2B). When liver or testis sections were stained with preimmune serum, no staining was observed (data not shown).

Fig. 2. Intracellulx localization of psly(ADP-ribose) synthetase. Sectisns of rat liver (A) and the PAP procedure. In @Q7 the lumen of seminiferous tubules (lm), interstitiai cells (in], sperm (apt), and round spermatids (spd) are indicated. (C) is a high magnifkalion of the testicular spermatocyt~s is indicated (arrov4 with spcf. The staining of spermatid cytoplasm is also shown in (C).

testis (B, C> were stained with antisynthetase antibody using (sp), spermatogonia (spg), Sertoli cdis (Ser), spermatocytes section. The strong staining of chromosomes in pachytenc in {C). Bars correspond to 100 pm in (A) and @), md 10 p-n

Poly(ADP-ribose) synthetase in spermatogenic cells

359

cm Fig. 3. SDS-polyacrylamide gel electrophoresis of the M-R fraction incubated with NAD. The isolated fraction was incubated with [14C]NAD (100 ~44) for 10 min, and the reaction was stopped with 10% trichloroacetic acid. The acid-insoluble material was washed once with the same acid, with acetone-O.1 N HCl and acetone, and the final residue was dissolved in SDS buffer. Aliquots were subjected to electrophoresis, and, after staining, the gel strips were cut transversely in 2-mm sections and processed as described under Materials and Methods. The large peak of radioactivity coincided with the boundary between the stacking gel and the separating gel. The molecular weights of standard proteins are indicated on the top. Migration was from left to right.

Automodification

of Poly(ADP-ribose) Synthetase

One of the most prominent characteristics of poly(ADP-ribose) synthetase is the ability to catalyze its own modification [2-51. As many as 15 chains with an average length of 80 ADP-ribose residues have been reported to be bound per molecule of enzyme [3]. This extensive modification of the synthetase forms a complex with a molecular weight in excess of half a million, which hardly enters polyacrylamide gels [4]. The activity associated with the M-R fraction synthesized oligomers with an average chain length ranging from 4.5 to 5.2 residues of ADP-ribose, which was similar to that of the products formed by the testis nuclei (about 6.4 ADP-ribose residues). The association of these oligomers with the synthetase in the M-R fraction was examined using polyacrylamide gel electrophoresis. As shown in Fig. 3, when M-R fraction was incubated with [14C]NAD and the products were analyzed by SDS-polyacrylamide gel electrophoresis, a single peak of radioactivity was recovered at the beginning of the resolving gel, which suggested ADP-ribose incorporation into a high-molecular-weight component, or apparently the automodified synthetase. A more direct proof of automodification of the synthetase came from a Western blot experiment (Fig. 4). The M-R fraction of nuclei of testis were incubated at 25°C with (or without) 100 @I4NAD, and the incubation mixtures were subjected to SDS-polyacrylamide gel electrophoresis. The separated proteins were then transferred to nitrocellulose [3 11, and poly(ADP-ribose) synthetase was localized

360 Concha et al. A

Fig. 4. Western blot analysis of poly(ADP-ribose) synthetase incubated with NAD. Rat testis nuclei (I, 2) and the M-R fraction (3, 4) were incubated with (1, 3) or without (2, 4) NAD. After incubation, aliquots were subjected to SDS-polyacrylamide gel electrophoresis, and the separated proteins were electrotransferred to two sheets of nitrocellulose. The first sheet was stained with the anti-synthetase antibody (A), while the second one was stained with Indian ink (B). On the left, the molecular weights of standard proteins are indicated.

with a specific antibody. In tracks 2 and 4 containing the nucIe fraction incubated without NAD, the antibody reacted with a molecular weight of 115,000 correspondi to the synthetase [3]. 0 stained bands of lower molecular wei s probably represented products of the enzyme during the incubation. In contrast, after incubation wit NAD, no band with the expected mobility of poly(A~P-ribose) synthetase was stained by the antibody (Fig. 4A, tracks 1 and 3). Instea , a diffuse staini noticed at the top of the gel, suggesting the for ation of a higb-rn~~ec~~a~-~ product. Since this material was recognized b the antibody cafthe synthetase, this result strongly suggested automodification of the enzyme, In agree previous work [2-51. After incubation with NAD, a drop in protein staining with Indian ink [39] was observed (Fig. 4B). The explanation of this drop is unknown at the present time. It can be argued that this is due to ADP-ribosylation of several proteins. However, this seems unlikely since one would expect a certain amount of smearing rather than less stained bands. DNA Requirement In previous experiments (Table 2), the synthetase was assaye of exogenous DNA. The addition of this component to the increased about threefold the activity associated with the The maximal activation was attained with about IO ygiml in activity was not due to synthesis of longer polymers of ADP-Chose, since the average chain length of oligomers formed in the absence and presence of was identical [40].

Poly(ADP-ribose) synthetase in spermatogenic cells

361

u Oib2b

DNA

(,,g/ml)

Fig. 5. Effect of DNA on the activity of poly(ADP-ribose) synthetase associated with the M-R fraction. The M-R fraction was assayed in the presence of varying amounts of calf thymus DNA, as indicated.

The residual or “DNA-independent” activity of poly(ADP-ribose) synthetase in the M-R fraction was suggestive of the presence of DNA in the preparation. Indeed, preliminary experiments showed that preincubation of the M-R fraction with DNase I led to a large decrease in the initial activity [24]. The fact that the DNase I effect was not due to inactivation of the synthetase by contaminating proteases but due to degradation of activator DNA was demonstrated by the experiment shown in Table 3. As previously observed, preincubation of the M-R fraction with DNase I for 6 h decreased the activity to 15% of the initial value. However, if the preincubation with the nuclease was carried out in the presence of 0.25 mM EGTA, an inhibitor of DNase I [41], no decrease of activity was noticed. Furthermore, a large proportion of activity lost by preincubation with DNase I was recovered by supplementing the reaction with calf thymus DNA (Table 3). These data strongly suggested the presence of DNA in the M-R fraction. The DNA component of the M-R fraction was then isolated with a phenol extraction procedure. Electrophoretic analysis of the DNA in agarose gel revealed a major component with a size similar to a large Hind111 fragment of i DNA (23,130 bp) (Fig. 6). Since the resolution at this region of the gel was poor, this DNA was TABLE 3 Effect of DNase I on poly(ADP-ribose) synthetase activity associated with the M-R fraction Preincubation conditions

Addition to assay

Activity (pmol/mg of protein)

Control +DNase I +DNase I+EGTA +DNase I

DNA+ EGTA

3222 543 3995 4559

Note. The M-R fraction isolated from testis of 30-day-old rats [24] was suspended in the reaction medium without NAD (see Materials and Methods) and preincubated for 6 h at 25°C in the presence of 250 pg/ml of DNase I, 0.2 mM PMSF, and 0.5 mg/ml of soybean trypsin inhibitor. After preincubation, the assay was carried out with 100 pA4 [14C]NAD (50 cpmipmol). The concentration of DNA and EGTA, where indicated, were 100 pg./ml and 0.25 m&f, respectively.

362

Concha et al. Eco RI ABAB

Barn HI AB

s

Fig. 6. Analysis of DNA isolated from the M-R fraction by agarose gel electrophoresis. Track A correspond to rat testis mtDNA and B to the DNA isolated from the M-R fraction of testis. The first two tracks on the left show native DNA, while the second and third pair indicate the fragments generated by EcoRI or BamHI digestion. S indicates MindIII fragments of i DNA.

further compared with DNA isolated from rat testis mitochon Fig. 6, the two DNAs were comparable in size. To ascertain DNAs were incubated with restriction enzymes, EcoRI and fragments generated were analyzed by electrophoresis ose gels. Essentiaiby treatment with ly identical fragments were generated from the two either EcoRI or BamHI (Fig. 6). That the DNA of the M-R fraction arose from contamination wit seems unlikely. The specific activity of s~c~inate-~ytoc~rome c reductase in the M-R fraction was l/lOth the activity found in the mito~h~~dr~al fraction of the testis (data not shown). In contrast, the yield of mtDNA isolate fraction was roughly similar to that from the mito~hondria~ fr more, the proportion of topoisomers in the DNAs isolated from the tw5 sub fractions was different: while about 70 % of the a was in a relaxed form, the DNA isolated fro several preparation of fraction was about 80% supercoiled. DISCUSSION

The present results, together with those of Agemori ef ~2. [38], that the testis has the highest specific activity of poly(ADP-ribose) synthetase compared with other organs in, at least, rat and mouse. This is true ind~~~rl~e~t~y if the activity (units of enzyme) is expressed in relation to milligrams of protein or

Poly(ADP-ribose)

synthetase

in spermatogenic

cells

363

of DNA. Being the synthetase a nuclear enzyme, the later form of comparison could be more adequate. However, the difference in activity (units/mg DNA) of the testis compared with the other organs should be also analyzed in the context of cell composition of the testis. In the adult rat testis the sperm and elongated spermatids, both rich in DNA, account for about 50% of the total cell population of the seminiferous tubules, while these cells are devoid of the enzyme activity (Fig. 2; [42]). Therefore, our results strongly suggest that spermatogonia and especially spermatocytes are quite enriched in the synthetase. In this regard, particularly appealing is the strong immunostaining of condensed chromosomes in pachytene spermatocytes (Fig. 2 C). In several cell types, a good correlation between terminal differentiation and a decreased poly(ADP-ribose) synthetase activity has been reported [43-45]. The present study indicated a similar situation to occur during spermatogenesis. The immunostaining of the enzyme increases markedly in spermatocytes in the meiotic prophase, then decreases progressively during spermiogenesis, and finally disappears in fully formed sperm. Recently, Corominas and Mezquita [46] have also reported that the content of poly(ADP-ribose) synthetase decreases during the differentiation of a germinal cell line of rooster. The significance of the high content of poly(ADP-ribose) synthetase in earlier spermatogenic cells is not clear at the present time. In this context, it is pertinent to recall the fact that the testis also contains the highest level of poly(ADP-ribose) glycohydrolase [47, 481. These results taken together strongly suggest that the metabolism of poly(ADP-ribose) may be an important biochemical event during spermatogenesis. Among other, there are two biochemical processes in which this metabolism could be involved. One is recombination at the meiotic prophase. Close relationship have been found between poly(ADP-ribosylation) of proteins and DNA repair, and between DNA repair and recombination [ll-131. This proposition agrees well with the strong immunostaining noticed in condensed chromosomes of pachytene spermatocytes (Fig. 2C) where the recombination event takes place. The other is reorganization of the genome during spermatogenesis, which involves relaxation as well as extensive condensation of chromatin 1231.There is good evidence for the participation of poly(ADP-ribosylation) of nuclear proteins in the modulation of chromatin structure [W-22]. In this regard, it is important to mention that ADP-ribosylation of histones and HMG proteins has been found in spermatogenic cells [49-5 11. Another interesting finding in the present study is the presence of poly(ADPribose) synthetase in the cytoplasm of spermatogenic cells, especially in round spermatids. Subcellular fractionation of the testis homogenate showed that a significant level of the synthetase activity was associated with the M-R fraction. This cytoplasmic localization of the enzyme was confirmed by immunocytochemistry using an antibody specific for this enzyme. A pertinent question is whether the enzyme immunolocalized in the cytoplasm is responsible for the activity associated with the M-R fraction. Besides the nucleus, only the M-R fraction contained a considerable level of enzyme activity. Furthermore, the synthetase activity was determined using the M-R fraction prepared from testis of rats at milligrams

v~ions periods after birth. It was found that the specific activi the rat testis M-R fraction reached a maxims value at 25 to ~Iermont and Perey [s2] have demonstrated a good chronological r~l~t~~n~~i~ between the age of rat and the type of terminal cells present in sern~n~~~r~ns tubules, Thus, round spermatids emerge at 25 to 3 days of age for the first ti and soon accoum for an important fraction of the s~ermatogeni~ cells. Since immnnostaining of the syntbetase was s~~ong~~ d clearer in the cyt round spermatids, the results indicate that the nativity as§o~iate~ with t fraction indeed ~o~espond to the immunolo~ali~e~ en~~rne~ fnn~tional meaning of this ~yto~Iasmi& poly(A l-0 spermatids is not clear at the present time. a~tivj%y ~onnd in the ribosomal fraction of nth~si~~d molecules of the enzyme [53], this seems -R traction of the testis. We have examined the 1 -R fraction isolated 2 h after the ~ntr~t~s~i~n~~~injection of ~~e~~~~~~~~~~~~. suits showed that the specific activity was not different from that of the Mh saline (data not s fraction isolated from control rats infected Another possible explanation for the presence of enzyme in the cylopl round spermatids is elimination of the enzyme fro of reorganization of haploid chromatin [23] e~~weve~~ this notion also appears to be improbable, since these structural changes take place mncb later in spermiogenesis [54]. Another possible role for the cy~op~asmi~ synt~e~ase is in the regn~atj~n of translation of mRNA. This possibility has been sng~est et aI. 1551.They fonnd poly(A~P-ribose) syntb~ta~e ac ity as§Qcia%edwith free cytsplasmic mRNP in mouse plasmacytoma cells. In matogenesis, there are good examples of mRNA which may be stored in t rm sf inactive m [X+%3], and poly(ADP-ribose) synt~etase might be inv~Ived in the or sn~~r~ssion of these messages. This attractive hesis warrants furt ies. The activity of poly(ADP-ribose) synthetase was stimulate by the addition of DNA, and the basal level of activity was de e presence of DNA in the M-R traction of the testis. DNA was identified as mitochondrial DNA. nation with mitochond~a seems unlikely. Pi of the M-R fraction revealed the absence of rn~to~~on~~ia~ ences in specific activity of succinate-cytochrome c reduc proportion of topoisomers of mtDNA between the two suggest that the DNA in the M-R fraction was n At the present time, however, the notion that t basal level of synthetase activity obser NA, such as heterogeered as tentative, because the presence ydisperse DNA, has not bee . To be mentioned in this ~~lydisperse DNA in human and boar sperm E591.

Poly(ADP-dose)

synthetase in spermatogenie cells

365

This research was supported by Grant RS-85-10 from the Research Fund of the Universidad Austral de Chile; Grant I/61 457 from the Stiftung Volkswagenwerk, FRG; Grants 186-87and 0942-88from the FONDECYT, Chile (LOB); and grants-in-aid for scientific research and cancer research from the Ministry of Education, Science, and Culture, Japan (K.U.).

REFERENCES 1. Ueda, K., and Hayaishi, 0. (1985) Annu. Rev. Biochem. 54, 73. 2. Yoshihara, K., Hashida, T., Yoshihara, H., Tanaka, Y., and Ohgushi, H. (1977) Biochem. Biophys. Res. Commun. 78, 1281. 3. Kawaichi, M., Ueda, K., and Hayaishi, 0. (1980) J. Biol. Chem. 255, 816. 4. Ferro, A. M., and Olivera, B. M. (1982) J. Biol. Chem. 257, 7808. 5. De Murcia, G., Jongstra-Bilen: J., Ittel, M. E., Mandel, P., and Delain, E. (1983)EMBO J. 2, 543. 6. Burzio, L. O., Riquelme, P. T., and Koide, S. S. (1979) J. Biol. Chem. 254, 3029. 7. Ogata, N., Ueda, K., and Hayaishi, 0. (1980) J. BioE. Chem. 255, 7610. 8. Yoshihara, K., Itaya, A., Tanaka, Y., Ohashi, Y., Ito, K., Teraoka, K., Masukage, A., and Kamiya, T. (1985) Biochem. Biophys. lies. Commun. 128, 61. 9. Jongstra-Bilen, J., Ittel, M. E.: Niedergang, C., Vosberg, H. P., and Mandel, P. (1983) Eur. J. Biochem. 136, 391. 10. Ferro, A. M., and Olivera, B. M. (1984) J. Biol. Chem. 259, 547. 11. Durkacz, B. W., Omidiji, O., Gray, D. A., and Shall, S. (1980) Nature (London) 283, 593. 12. Jacobson, E. L., Entol, K. M., Juarez-Salinas, H., and Jacobson, M. K. (1983) J. Biol. Chem. 258, 103. 13. Wielckens, K,, George, E., Pless, T., and Hilz, H. (1983) .I. Biol. Chem. 258, 4098. 14. Farzaneh; F., Zalin, R., Brill, D., and Shall, S. (1982) Nature (London) 300, 362. 1.5. Althaus, F. R., Lawrence, S. D., He, Y. Z., Sattler, G. L., Tsukada, Y., and Pitot, H. C. (1982) Nature (London) 300, 366. 16. Tanuma, S., and Johnson, G. S. (1983) J. Biol. Chem. 258, 4067. 17. Slattery, E., Dignam, J. D., Matsui, T., and Roeder, R. G. (1983) J. Biol. Chem. 258, 5955. 18. Kurl, R. N., and Jacob, S. T. (1985) Nucleic Acids Res. 13, 89. 19. Butt, T. R., Jump, D. B., and Smulson, M. E. (1979) Proc. Natl. Acad. Sci. USA 76, 1628. 20. Burzio, L. O., Koide, S. S., Puigdomenech, P., and Ruiz-Carrillo, A. (1980) N6vel ADPRibosylation of Regulatory Enzymes and Proteins (Smulson, M. E., and Sugimura, T., Eds.), p. 34.5,Elsevier, New York. 21. Poirier, G. G., De Murcia, G., Jongstra-Bilen, J., Niedergang, C., and Mandel, P. (1982) Proc. Natl. Acad. Sci. USA 79, 3423. 22. Singh, N., Poirier, G., and Cerutti, P. (1985) EMBO J. 4, 1491. 23. Bellve, A. R., and O’Brien, D. A. (1983) Mechanism and Control of Fertilization (Hartmen. J. F., Ed.), p. 35, Academic Press, New York. 24. Concha, I. I., Concha, M. I., Figueroa, J., and Burzio, L. 0. (1985) ADP-Ribosylation of Proteins (Althaus, F. R., Hilz, H., and Shall, S., Eds.), p. 139, Springer-Verlag, Berlin. 25. Laemmli, U. K. (1970) Nature (London) 227, 680. 26. Vera, J. C., Brito, M., Zuvic, T., and Burzio, L. 0. (1984) J. Biol. Chem. 259, 5970. 27. Tishler, P. V., and Epstein, C. J. (1968) Anal. Chem. 22, 89. 28. Fox, B. W. (1976) Laboratory Techniques in Biochemistry and Molecular Biology (Work, T. S., and Work, T. S., Eds.), p. 41, North-Holland, Amsterdam. 29. Ikai, K., and Ueda, K. (1980) Biochem. Biophys. Res. Commun. 97, 279. 30. Ikai, K., and Ueda, K. (1983) J, Histochem. Cytochem. 31, 1261. 31. Tsang, B., Peralta, J., and Simons, A. In Methods in Enzymology (Langone, J., and Van Vunakis, H., Eds.), Vol. 92, p. 377, Academic Press, New York. 32. Stemberger, L. A., Hardy, P. H,, Cuculis, J. J,, and Meyer, H. G. (1970) J. Histochem. Cytochem. 18, 315. 33. Van Tuyle, G. C., and McPherson, L. (1979) J. Biol. Chem. 254, 6044. 34. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 35. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265. 36. Shima, T., Hasegawa, S., Fujimura, S., Matsubara, H., and Sugimura, T. (1969) J. Biol. Chem. 244, 6632. 37. Giles, K. W., and Myers, A. (1965) Nature (London) 206, 93. 24-898332

366 Conch et al. 38. Agemori, M., ~ag~iyama, II., Nishjk~mi, M.. and Shizuta, Y. (1982) Arch. Biochein. Biophys. 621. 39. cock, K., and Tsang, V. C. W. (1983) Ad. ~iuc~ei~. P%, 157. 40. Burzio, L. O., Concha, I. I., Figueroa, J., and Concha, M. I. (1983) A~~-~~b~syIat~~~~ DNA Repair and Cancer (M&a, M., ~aya~sh~, O., Shall, S,, ~rnu~so~,M. E., and ~u~~~r~~ ‘P.~Eds.), p 141, Scientific Societies Press, Tokyo. 41. ce, P. A. (1975) J. Bid. Chem. 250, 1981. 42. ‘0; L. Q., Saez, L., and Cornejo, R. (1981) ~~oc~e~. Bio;nliys. Ref. Cummzm. 1 43. ous, J. W., Furneaux, H. M., Pearson, C. K.; Lake, C. RI., and Morrison ~joc~le~. 1. 180, 455. 44. Ikai, K., Danno, K., Imamura, S., and Ueda, K. (1982) .?. Dermatol. 9, 125. 45. Qhashi, Y., Ueda, K., Hayaishi, Q., Ikai, Pa,,>and Niwa, 6). (1984) Proc. I\irrtl. Acad. Sci, lJS% 81, 7132. 46. Corominas, M., and Mezquita, C. (1985) J. Bid. Chem. %Q, 16,269. 47. Miwa, M., N~atsuga~, K., Hara, K., ~ats~sb~ma, T., and ~~girn~~, T. (1975) Ardz. Obochc~ra. 7. 167, 54. L. Q., Riquelme, P. T., Clhtsuka, E., and Koide, S. S. (1976) Arch. Biochem. Blcpjhvs. 6. 49. Burzio, L. Q. (1982) ADP-Ribosyla~on Reactions: Biology and ~cdic~n~ (Heyaishi, 0.. and Ueda, K., Eds.), p. 103, Academic Press, New York. 50. Wong, N. C. W., Poirier, 6. G., and Dixon, G. (1977) Eur. 9. Biochem. 77, II. 51. ~~o~~-~ennella, M. R., Quesada, P., Farina, B., Leone, E., and Jones, R. (1984) .%&em. J. 224, 223. 52. Ckrmont, Y., and Perey, B. (1955) Amer. J. Anat. ‘B 53. Roberts, J. H., Stark, P., Ciri, C., and Smuison, M. rch. Biochem. &phq‘s. 171, 305. 54. Grimes, S. R,, Meistrich, M. L., Platz, R. D., and Hnika, L S. (1977) Exp. Cdl 55. T~omass~n, II., Niedergang, C., and Mandel, P. (1985) &&hem. Biophys. Res. Co~nmutz. 133, 554. 56. Gold, B., Fujimoto, II., Kramer, J., Erickson, W., and He&, N. (1983) Dev. B&i. 98, 392. 57. Iatrou, K., Spira, A., and Dixon, 6. (1978) L)eu. Rioi. 64, 82. 58. Sinclair, A., and Dixon, G. (1982) ~~oc~e~~§tly 21, 1869. 59. McGrath, J. P., and Evenson, D. P. (1982) Gamete RES. 5; 379. Received April 19, 1988 Revised version received September 5, 1988