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Radiation-initiated DNA synthesis in spermatogenic cells of the mouse
Experimental
Cell Research 69 (1971) 33-44
TATION-INITIATED DNA SYNTHESIS I CELLS OF THE SUSANA KOFMAN-ALFARO
and ANN @. CMAN
Medical Research Council, Clinical and Population C’ytogenetics Unit, Western General Hospital, Crewe Road, Edinburgh 4, Scotland
SUMMARY 1. “Unscheduled” DNA synthesis has been demonstrated in spermatogenic celis of the mouse following high and low doses of UV and high doses of X-rays, using autoradiographic techniques. 2. A small amount of “natural” DNA synthesis also occurs at the zygotene/pachyteae stages of meiotic prophase. 3. Little or no “unscheduled” DNA synthesis can be detected in testicuiar spermatozoa, but spermatogonia, spermatocytes and spermatids show varying levels of synthesis. 4. Maximum levels of “unscheduled” DNA synthesis are found in late-zygotene/ear?y pachytene spermatocytes, the cell-stage showing some “natural” DNA synthesis in the controls. 5. The results are discussed in terms of a general “repair” system operating in spermatogenic
The detection, by autoradiographic means, of a low level of DNA synthesis following UV irradiation of HeEa cells in all stages of the cell cycle, was first made by Rasmussen & Fainter [I]. This radiation-stimulated synthesis has been termed “unscheduled synthesis” [I!] and has since been found in a variety of other mammalian cell types following both UV and X-n-radiation [3,4]. Caesium chloride density centrifugation studies have shown that the stimulated incorporation of labelled precursor into 6)NA following irradiation of mammalian cells shows features in common with ‘“repair rephcation” observed in bacteria [S, 41. Repair replication occurs at random points along the DNA chain, is non-semiconservative, and in bacteria, is part of a repair process (excision repair), that results in enhanced survival [7]. It is believed that “unscheduled synthesis” and “repair replication” are manifestations 3 - 711818
of the same basic “repair” process HeLa cells, “unscheduled syn been observed after low doses of tion that result in high survival, and it is thus thought that nearly all cells in such populations perform repair replication an retain their reproductive integrity [3]. That the “repaired” DNA of human cells can subsequently undergo normal replication has been shown by Rasmussen et al. [9] and Painter et al. It is well k n that individual stages of oogenesis and spermatogenesis in a variety of organisms vary widely in their se~s~t~v~t~es to irradiation and other agents in terms of cell-killing [I l-141, mutation induction [iZ, 15, 161, and chromosome breakage [97], and while the final radiosensitiv germ-cell stage appears to a variety of factors, the differential capacity of each stage to repair damaged DNA is Exptl Ceil Res 69
34 SusanaKofman-Alfaro & Ann C. Chandley considered to be one of prime importance [16, 18, 19, 20, 211. Evidence from bacteria [22] and Paramecium [23] has shown that repair of pre-mutational damage is invariably associated with DNA synthesis, and we considered it of interest to look for direct evidence of DNA synthesis following irradiation of mammalian germ cells. The present paper gives information from the mouse; our findings for human germ cells will be the subject of a subsequent publication [24]. METHODS The male mice used in the experiments were from a random-bred Q strain and were 6-8 weeks old at the time of killing. Irradiation and 3H-thymidine labelling were carried out in vitro on cellular suspensions of testicular cells prepared by two different methods: (1) For UV irradiations, testes were chopped with fine scissors in sterile saline, until a suspension of germ cells was obtained. The suspension contained all spermatogenic stages from spermatogonia to testicular spermatozoa. In the first series of UV experiments, 1 ml of suspension was irradiated with 2 537 8, light from a low pressure mercury lamp (‘Hanovia’ type) at a doserate of 1 400 ergs/mm2/min in a plastic Petri dish of 3.5 cm diameter. A total dose of 4 200 ergs/mm2 was given. An equal part of suspension formed the unirradiated control. In a subsequent series of experiments, suspensions were irradiated at a dose-rate of 4.2 ergs/nm?/sec to give total doeses of 50 and 100 ergs/mm2. Unirradiated control suspensions were also prepared. Immediately after irradiation, both treated and control suspensions were centrifuged at 1 500 rpm for 5 min, the cells resuspended in 2 ml Eagle MEM (supplemented with 20 % calf serum, 200 IU/ml penicillin and 200 ,ug/ml streptomycin) containing 10 @Zi/ml 3H-thymidine (spec. act. 22 Ci/mmole), and incubated for 2 h at 37°C in a gas phase of 5 % CO, and 95 % air. After incubation, the suspensions were again centrifuged, the supernatant discarded, and the cells resuspended in hypotonic sodium citrate (1 X) for 15 min prior to fixation in 3 : 1 methanol : acetic acid. They were then processed according to the method of Evans et al. 1251and air-dried preparations made. Two replicate runs of the experiment-were performed at the high dose of UV i.e. 4 200 ergs (Runs R2 and R7), and one run at each of the low doses i.e. 50 and 100 ergs (Runs R8 and R9 respectively). (2) For the X-ray experiments, half of each of the right and left testes were exposed to a dose of 5 000 rad in sterile saline, while the other halves were kept as unirradiated controls. Both treated and control Exptl
Cell Res 69
pieces were then incubated for 2 h at 37°C in 5 ml Eagle MEM containing 15 ,&i/ml 3H-thymidine (spec. act. 22 Ci/mmole). After washing in nonradioactive medium, they were transferred to hypotonic sodium citrate (1 %) for 15 min, and then chopped with fine scissors until a suspension of cells was obtained. Air-dried preparations were then made as above [25].
Autoradiography All slides were stained with carbol fuchsin [26] and filmed for autoradioarauhv bv dinning in Ilford L4 liquid emulsion. Adequate au&radiographs were obtained following UV irradiation after a 2 week exposure period. A period of 6 weeks was necessary for cells irradiated with X-rays. Additional slides from each experiment were exposed for 34 months. Some slides from each exneriment were treated with DNAase for 1 h at 37°C in 0.1 M Tris-HCl (pH 7.0) with 0.2 M MeCL which contained 10 r&ml DNAase. Controls were incubated under the same conditions in buffer solution alone. Slides were then washed in distilled water, extracted in 1 % perchloric acid at 4°C for 20 min and washed again.
Definition of cell stages In the testicular suspensions obtained, all stages from spermatogonia to spermatozoa could be recognised. The identification of individual stages was based on our own previous studies of meiosis in the male mouse [27] and on descriptions given by Oakberg
1281.
Treatment of cells by the two different techniques produced slight differences in the final distribution of cells in suspension. For example, fewer spermatogonia, resting primary spermatocytes and leptotene nuclei were seen in suspensions prepared by Method 2 than in those prepared by Method 1. Cells in diplotene were rare in suspensions prepared by Method 1 and absent in those prepared by Method 2. Cells in diakinesis were extremely rare in both.
RESULTS High Dose UV and X-ray Experiments
Unscheduledsynthesis in spermatogoniaand resting primary spermatocytes In control cultures of both the UV and X-ray experiments, heavily-labelled spermatogonia and resting primary spermatocytes, which were in S phase at the time of label incorporation, were found (table 1). These showed various replication patterns depending on the time in the S period at which incorporation had occurred [27].
Unscheduled DNA synthesis in mouse gem cells
35
Fig. 1. Autoradiograph of testicular cells of the mouse following irradiation with a UY dose of 4 200 ergs/mm” and incubation in medium containing 10 &i/ml 3H-thymidine for 2 h. Heavily labelled S period spermatogonia contrast with lightly-labelled late zygotene/early pachytene spermatocytes undergoing unscheduled DNA synthesis. (a) Mid-S pattern (b) end-of-S pattern.
After irradiation with 4 200 ergs/mm2 or 5 000 rad X-rays, two distinct populations of labelled spermatogonia and resting primary spermatocytes were seen. Approximately half the labelled cells were heavily-labelled and were interpreted as S phase cells while the other half were lightly-labelled, and were interpreted as non-S-phase cells showing ‘“unscheduled” DNA synthesis (table 1, fig. 11. ean gram counts per nucleus (based on labelled cells only) for lightly-labelled cells in the two runs of the UV experiment were 2S.Ori: 1.5 and 33.313.1 respectively and the mean for the X-ray experiment was 25.3 i 3.0. Heavily labelled S phase cells in control cultures showed higher levels of incorporation of label than those seen in irradiated cultures. This was particularly obvious following UV irradiation, and it was concluded that some inhibition of S period DNA synthesis had
Table 1. S period and radiation ir?duced “unscheduled” DNA synthesis in spevmatogonia and resting primary spermatocytes following high doses of UV and X4~adiation
No. of cells Run
Control
UV 4 200 ergs R2 100 R7
X-Ray 5000 rad
100
5Q
Treated
No. labelled Cortrol Treated ___ 3% 3% 2 ‘2 3 ‘2
100
1
100
0
100
2
45 62
12
47 40
4i
34
24
31
Low = lightly-labeiled cells performing unschedu?ed DNA synthesis. High = heavily-labelled cells performing S period DNA synthesis.
36
Susana Kofman-Alfaro
& Ann C. Chandley
Table 2. Grain count estimates for various meiotic stages following 4 200 ergs UV irradiation and in controls No. of cells
% labelled
Cell stage
Control
Treated
Control
Treated
Control
Treated
Leptotene a b Zygotene a b Late zygotene/ early pachytene a b Late pachytene a b Diplotene a b 2” Spermatocyte a b Round spermatids a b Elongating spermatids a b Spermatozoa a b
150
200 -1 300 -
2.0 2.7 0.7 9.6
34.0
1.2kO.3 3.6kO.4 1.3kO.2 5.3 kO.4
8.0f1.3
300
6.7 49.0
Autoradiographic 1 Not scored.
75 300 250 300 300 300 250
GO
cpm
53.3 98.5 87.7
1::; 2:::
GO -.
i:
30
200 150 200 150 200 200 200 200
200
0.5
71.0
100
9.0
91.0
500 150 300 300 300 500
0 5.5 0 1.5 0 0
46.6 82.0 2.7 30.0 i.4
3.3 +0.4 13.720.6 2.OkO.3 6.6kO.4
17.3 +2.1 71.213.5 46.8 +3.4
2.4kO.8 7.8kO.9
41.7T8.7 -
0.9F0.2 2.8kO.2 0.5kO.l 2.7i0.2 0.2110.07 0.77+0.09 0.1010.06 0.28iO.05
13.7k1.6 59.Ok3.7 6.2kO.5 40.0i2.6 0.88kO.24 4.51kO.31 0.14+0.04 1.43 10.09
exposure period: (a) 2 weeks (b) 34 months.
occurred following UV irradiation, a phenomenon which has been extensively reported in previous publications [l, 291. The high level of S period labelling in both control and treated groups precluded grain count estimates from being made. Treatment with DNAase resulted in an almost complete disappearance of grains from all cells, showing that label was actually in DNA.
cells were considered to be those having significantly higher counts than the background count for an area of equal size on the slide. In control cultures, after two weeks exposure to the autoradiographic film, a small non-significant increase in incorporation of 3H-thymidine was found in late zygotene/early pachytene spermatocytes relative to other stages (table 2, fig. 2). In slides exposed for 34 months, this peak of Unscheduled synthesis in spermatocytes, labelling was more apparent, the mean count spermatids and spermatozoa per cell at this time being 13.7+ 0.6 (table 2, Grain count estimates for primary and fig. 2). A t test showed this to be signifisecondary spermatocytes, spermatids and cantly higher than the pooled means for spermatozoa following a high dose of UV zygotene (5.3kO.4) and late pachytene irradiation and in their controls are given in (6.6rt: 0.4) (t = 11.27) (P=O.OOl). table 2 and fig. 2. Patterns of labelling are This small peak of “natural” DNA synthesis in the controls corresponds, we believe, shown in fig. 3. Mean counts per cell were based on the to the small amount of prophase DNA total cells scored for each individual stage synthesis which has been claimed to occur i.e. labelled and unlabelled cells. “Labelled” in spermatocytes of the newt [30] and has Exptl Cell Res 69
own biochemically, to occur in 89 rocytes of kilium and Trillium at these stages [31]. (In a recent publication 1231 we reported our failure to detect prophase NA synthesis in male mice injected with TI-thymidine, and concluded this was due to the poor resolution afforded by the auto60 radiographic technique. We now believe that it was due to the fact that insufficient radioactive isotope wa aken up by prophase cells in the testis. th the present in vitro technique, cells have greater opportunity to incorporate label and detection of even low NA synthesis becomes possible.) exposed to a dose of 4 200 -tbymidine incorporation was detected in all spermatogenic stages with the exception of late spermatids and spermatozoa. No incorporation of label was observed in Sertoli cells. Since the meiotic S period in the mouse occurs at preleptotene in the resting primary spermatocyte this incorporation cannot be attributed to normal NA synthesis, and is thus a radiation-stimulated “‘unscheduled” synthesis comL 2 LZiEP Li= D parable to that found in irradiated somatic cells. Significant depressions in grain count were seen after treatment with DNAase, Fig. 2. Abscissa: spermatogenic stage; ordinate: mean 2 weeks autora?iographic grain count/cell. -, showing that the label was in fact in DNA. exposure. ---, 34 months exposure. Among primary spermatocytes, almost Grain count estimates for individual germ-cell stages following irradiation, with a UV dose-of 4 200 10 % of cells in late zygotene/early pachytene ergs/mm2 (T) and in controls (C). L, leptotene; Z, showed an incorporation of 3H-thymidine zygotene; LZIEP, Iate zygotenejearly pachytene; Lk, late pachytene; D, diplotene; 2” Cyte, secondary which far exceeded that of any other prophase spermatocyte; R. tid, round spermatid; E. tid, elongatstage (table 2, fig. 2)~ The mean count per ing spermatid; SF, Spermatozoa. ceb for late zygotene spermatocytes (71.2 k few cells in diakinesis/metaphase I were seen 3.5) was nearly twice that of the next most to make scoring of this stage feasible, of heavily-labelied stage i.e. late pachytene those that were seen, a few (4 out of 16) (40.8 + 3.4), and nearly ten times higher than were labelled with counts ranging from IO-30 the mean for leptotene (S.Oi: 1.3). There was no significant difference in the grains, while the others were un‘iabelied. The mean grain counts for secondary mean grain counts for late pachytene and spermatocytes and round spermatids were diplotene. The large standard error for and 6.22 .5 respectively, thus diplotene was presumed to be due to the 1X7&1.6 showing roughly equal levels of unscheduled low numbers of cells scored. Although too
38 SusanaKofman-Alfaro & Ann C. Chandley
Fig. 3. Unscheduled DNA synthesis in various stages following irradiation. All cells shown are from UV-treated cultures (4 200 ergs/mmz) exposed to film for 2 weeks with the exception of mid-pachytene spermatocytes shown in fig. 3~. These were from X-rayed cultures (5 000 rad) exposed to film for 6 weeks. (a) Zygotene; (b) late zygotene/early pachytene; (c) mid-pachytene; (d) late pachytene; (e) diplotene; (f) diakinesis; (g) 2” spermatocyte; (Jr) round spermatids. Exptl
Cell Res 69
Fig. 4. Autoradiograph of unirradiated control cells showing a low level of “natural” DNA synthesis at the late zygotene/early pachytene stage of meiotic prophase. Autoradiographic exposure period 6 weeks.
iW content, whereas the values for elongating spermatids and spermatozoa were 9.88 +_9.24 and 9.14 + 0.94 respectively. In UV-treated cultures exposed to film for 3$ months, primary spermatocytes were so heavily labelled that accurate grain counting was not possible. The levels of unscheduied DNA synthesis in elongating spermatids and sperm were stiff extremely low, the mean counts for these stages being4.51 k 0.31 and 1.43 i- 9.09 respectively (table 2, fig. 2). In. view of the condition of exposure, it seemed highly unlikely that the absence of significant Iabelling in elongating spermatids and spermatozoa exposed to UV was due to failure of UV penetration. However, in order to clarify this point, the cells were exposed to 5 000 rad X-rays.
The results for a single run of t experiment are given in table 3 and fig. 5. After an exposure period of 6 weeks the control data again showed a small but significant peak of “‘natural” DNA synthesis in late zygotene/early pachytene spermatocytes, the mean count being 5.419.4 grains per cell compared with counts of 2.25 9.2 and 2.0 2 0.2 for zygotene and mid-pachytene respectively and 0.8 5 0.2 for teptotene. After 34 months exposure (table 3), the mean count per cell for late zygotenelearly pachytene was 49.3 F9.9 which was significantly higher than the pooled means for zygotene (3.7 * 0.5) and mid-pachytene (3.4 I 0.5) (t=6.75; P= .OOZ). The results for the controls of both the UV and X-ray experiments thus indicate that natura’f DNA
40
Susana Kofman-Alfaro
h Ann C. Chandley
Table 3. Unscheduled DNA synthesis following 5 000 rad X-rays No of cells
% labelled
Stage
Control
Treated
Control
Leptotene a
41 50 200 100
100 -1 150
0 2.0 3.5 8.0
300 100 150 100 150 50
150 150 100 -
16.0 29.0 2.0 6.0 0 0
150 100 150 100 100 100 100 100
300 100 300 100 200 200 200 200
0 3.0 0 1.0 0 0 0 0
b Zygotene a b
Late Zygotenej Early pachytene a b
Mid pachytene a b Late pachytene a b
2” Spermatocyte a b
Round spermatid a b
Elongating spermatid a b
Spermatozoa a b
Autoradiographic l Not scored.
Cell Res 69
Treated 3.0
47.3 73.3 ii.0 -
8.0
18.3 25.0 19.7 42.0 0.5 8.0 0 0.5
Control
Treated
0.8 io.2 1.6iO.4 2.210.2 3.7kO.5
1.3 +0.4 14.321.3 -
5.420.4 10.3 LO.9 2.oio.2 3.6kO.5 1.0~0.1 1.9kO.4
37.7 k2.5
0.6 LO.1 1.220.2 0.5 +0.1 1.7iO.2 0.24 kO.05 0.37 kO.08 0.12+0.03 0.21 kO.05
9.6T1.3 4.5i1.3 3.3 jo.3 7.151.1 3.3 +0.3 lO.Oi1.3 0.15 io.05 1.56f0.21 0.07 10.02 0.40 10.09
exposure period: (a) 6 weeks; (b) 39 months.
synthesis occurs throughout the zygotene and pachytene stages of prophase reaching a peak at late zygotene/early pachytene. No significant prophase synthesis occurs at leptotene. The results for the X-irradiated cultures after an exposure period of 6 weeks confirmed our main findings with UV, showing (i) late zygotenelearly pachytene was the stage at which maximum levels of 3H-thymidine incorporation occurred, and (ii) elongating spermatids and spermatozoa showed little, if any, incorporation (table 3, fig. 5). (iii) No incorporation was seen in Sertoli cells. The mean grain count for late zygotene/early pachytene after 6 weeks’ exposure of the films, was 37.7k2.5 (approximately half that obtained with UV after only 2 weeks’ exposure). After 3& months exposure primary spermatocytes in the X-rayed cultures showed levels of incorporation which were too high for accurate grain estimates to be made. As in Exptl
cpm
the UV experiments elongating spermatids and spermatozoa showed negligible incorporation (table 3, fig. 5). Low Dose UV Experiments
The UV dose of 4 200 ergs/mm2used in the first experiments was almost certainly lethal to the cells exposed. The question arises therefore, whether the labelling observed is part of a pathological process, leading to cell death. Clarkson & Evans (personal communication) in our laboratory have shown that a maximum incorporation of label occurs in human lymphocyte DNA after exposure to 40 ergs. Mouse germ cells therefore were exposed to doses of 50 and 100 ergs/mm2. Selected stages at each level of ‘ploidy’ were scored and the results are given in table 4 and fig. 6. The results show that unscheduled synthesis is clearly evident following low dose exposures.
Table 4. Unscheduled
NA synthesis in ceh exposed to UT/ doses QJ”50 and i&3 ergslmna2 -Dose No. % Stage
mm2
Zygotene
50 100 Late zygotene/ 50 Early pachytene 100 2” Soermato&e Round spermatids
RXd
ETid
Labelled
cpm
-
50 50 50
44.0 42.0 100.0
9.9k1.5 14.1 k2.5 46.8 -c 3.0
50
96.0
54.5*4,1
50 IO0
5 50
58.0 66.0
7.9 jo.9 14.1 xl.7
50 100
100 50
64.0 58.0
Autoradiographic
‘Cyte
Cells
6.8 iO.6 8.6&1.1
exposure period 2 weeks.
Sp
4c------l~zc+r----cDNA CONTENT
Fig. 5. Abs&8ssa: spermatogenic stage; ordinate: mean grain count/cell. -, 6 weeks autoradiographic exposure; ---, 34 months exposure. Grain count estimates for individual germ-cell stages following irradiation with 5 000 rad X-rays (T) and in controls (C). Abbreviations as in fig. 2. MP, mid-pachytene.
Localization of DNA Synthesis
The possibility existed that some of the NA we were observing could have been in the cytoplasm closely surrounding the nucleus and not in the nucleus itself. That this was not so, was evident from slides stained with the fluorescent stains acridine orange and “Atebrin”, which showed that most of the nuclei in the preparations were naked and free of cytoplasm. Cytoplasmic Pabelling was however found around many human spermatogenic cells [24] and when these were stained with “Atebrin” it was clear that the cytoplasm still adhered to most of the nnciei. Why cytoplasm should be shed from mouse cells but not from human cells, when the technique used in both cases is the same, is unknown.
Z w DNA CONTENT
LZiEP 4c~-Pc+------C--------d
-cyte
RTid
Fig. 6. Grain count estimates for selected germ-cell stages following irradiation with high and low doses of UV, -, 4 200 ergs/mm2; ---, 100 ergs/mm2; -.-.-., 50 ergs/mm2. Exposure to autoradiographic film 2 weeks. Abbreviations as in figs 2 and 5.
42
Susana Kofman-Alfaro
& Ann C. Chandley
DISCUSSION We have demonstrated that “unscheduled” DNA synthesis is initiated in spermatogenic cells of the mouse following exposure to UV or X-rays and that the level of stimulated synthesis varies from one germ-cell stage to another. Furthermore we have found a similar pattern of response in germ cells subjected to “high” and “low” doses of UV and to “high” doses of X-rays, suggesting that the same basic process may be operating at all dose levels and with both kinds of radiation. Whether this stimulated synthesis is part of a repair process that restores damaged DNA to its original state is not clear. In its broadest sense, the term “repair” applied to irradiated meiotic cells could be considered in terms of enhanced survival, reduced mutation frequency (“repair of premutational damage”) and should also include events resulting in chromosome exchange and possibly the normal process of exchange, associated with crossing-over. If “repair” of DNA is involved in all these phenomena, there is no evidence that this “repair” is the same for all these events or even that common mechanisms are involved. However, evidence from bacteria has revealed an association between recombination and repair of UV-induced damage. Mutants of E. coli K12 have been isolated which are unable to form recombinants by conjugation (Ret-) and are also unable to repair photodamaged DNA [32, 331 and it has been suggestedthat certain enzymes and common pathways may serve both in genetic recombination and in repair following irradiation [34]. In eukaryotes there is also some evidence that recombination-deficient mutants may be radiation-sensitive. Watson [35] has shown, for example, that the recombination-deficient c3G of Drosophila melanogaster is more radiation-sensitive than wild-type flies in Exptl
Cell Res 69
terms of dominant lethal induction. Also the data of Riley & Miller [36] suggeststhat a desynaptic mutant in barley is more radiation-sensitive in terms of aberration yield than the wild type, although confirmation of this could not be obtained in more extensive experiments carried out by Swietlinska & Evans [37]. Finally in man, a patient showing a desynaptic condition in most spermatocytes at diakinesis has been found to show reduced levels of “unscheduled synthesis” in the blood [37a]. It has been suggested 138, 391 that the mechanisms involved in the exchange which occurs at meiosis to give chiasmata and crossing-over may be the same as those leading to the formation of chromosome aberrations. If our unscheduled synthesis is indeed part of a “repair” process operating at meiosis, some interesting correlations with the genetic data on repair in germ-cells can be seen.It is known from work on Drosophila [40, 411 the mouse [18, 191 and sea urchin [42] that sperm show no repair, following irradiation, until fertilization. Other gametogenie stages, e.g. spermatogonia, spermatids and oocytes, are assumed to undergo some repair on the basis of evidence from doserate and other experiments [18, 19,431. If our unscheduled DNA synthesis in the mouse is a manifestation of “repair”, then our results would support the genetic finding of “no repair in sperm” by showing little or no unscheduled synthesis in them. The lack of repair in sperm may be due to their low metabolic activity [18, 211 or may be a consequence of the fact that the DNA in sperm is tightly packaged in basic protein and may not be accessibleto repair enzymes. If the “repair capacity” of different germcell stagesis associated with intrinsic factors such as metabolic activity, the finding of maximum levels of unscheduled DNA synthe-
sis in late zygotene/early pachytene spermatocytes may be associated with a high metabolic activity. The enormous growth of spermatocytes from leptotene to pachytene suggestsincreasing metabolic activity. One of the most striking findings in our experiments is a highly significant peak of -thymidine incorporation at late zygotene/ early pachytene in irradiated cells and a coincident peak of “‘natural” DNA synthesis in the controls. “‘Natural” prophase DNA synthesis has also been found to occur in microsporocytes of Lilium and Trillium at the zygotenej pachytene stages[31,44,53] and this synthesis has recently been analysed by the techniques Of A-DNA hybridisation, density-labellin nd by the use of the DNA synthesis inhibitor hydroxyurea [53]. Zygotene synthesis in Eilium appears to represent a delayed semiconservative replication of approx. 0.3 Y0 NA and shows a higher G + C lk nuclear DNA. Its function in meiosis appears to be associated with chromosome pairing [64]. DNA synthesised during pachytene represents approx. 0.1% of the genome and appears to have the characteristics of a “repair replication” [53]. The ilure of bromodeoxyuridine-substituted NA synthesisedduring pachytene to show a “‘heavy shift” in buoyant density and the ineffectiveness of hydroxyurea in arresting pachytene DNA synthesis are taken as evidence to support this conclusion. Pachytene NA synthesis is believed to be associated with the exchange process resulting in crossing-over c45, 531. Although there is no direct evidence that prophase synthesis of DNA is involved in crossing-over its occurrence has come to be associated with those hypotheses which postulate a “repair’‘-type NA as the terminal event in the crossing-over process [46, 471. Indirect evidence has now accumulated which suggests
that a critical step in the crossing-over process does occur at the zygotenejpac meiotic prophase [48, 49, 501 step involves the synthesis of Furthermore, owe11 6% Stern [55] have recently identified a set of ~Qrn~Ie~~~tary d repair enzyme activities
believe may sponsor a breakage-reunion mechanism responsible for genetic recombiur findings of coinci NA synthesis a initiated” “ unscheduled” synthesis in mouse spermatocytes at late zygotenelearly pachytene correlate well with the increased endonuclease, polynucleotide kinase and ligase activities found during this stage in kllilam [55], and if ow unscheduled in the mouse represents t damaged NA our results may indicate common enzymic activities in the phenomena of breakage, excision and exchange in recombination and in t e repair of UVdamaged DNA. If at least a part of the ““natural” synthesis occurring at prophase in the is a semi-conservative replication similar to that occurring during the S period, we might have expected inhibition of this synthesis following irradiation. Inhibition of NA synthesisfollowing irradiation has been uE.d in S phase cells of the testis and in S phase somatic cells [l, 3, LB]. On the contrary, our results showed an enormous increase in incorporation of label into the DNA of late zygotenelearly pachytene spermatocytes-the stage at which “natural” DNA synthesis was found. This amounted to an g-fold increase in X-rayed cultures and a 25-fold increase in UV-treated cultures as compared with controls. The amount of stimulated synthesis observed at this time is als greater than the unscheduled
44 SusanaKofman-Alfaro & Ann C. Chandley induced in cells in other stages of meiosis at the time of irradiation. It seemsprobable therefore that the large amount of radiation induced synthesis at zygotene/pachytene may reflect the involvement of a recombinational repair process that is normally operative, at a much reduced level in ‘control’ cells at this time. Unscheduled synthesis at the other meiotic stages may be more comparable to that observed in somatic cells. The authors are grateful to Professor H. J. Evans for his help and advice during the course of this work, and for his criticism of the manuscript. They also wish to thank Mrs Judy Fletcher for excellent technical assistance and Mr Norman Davidson for his help in the preparation of the photographs.
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I
395.‘
8. Painter, R B & Cleaver, J E, Rad res 37 (1969) 451. 9. Rasmussen, R E, Reisner, B & Painter, R B, Int j rad biol 17 (1970) 285. 10. Painter, R B, Umber, J S & Young, B R, Rad res 44 (1970) 133. 11. Cocchi, U and Uggeri, B, Strahlentherapie 75, (1944) 96. 12. Russell, W L, Radiation biology (ed A Hollaender) vol. 1, p. 825. McGraw-Hill, New York (1954) 13. Oakberg, E F, Rad res 2 (1955) 369. 14. Mandl A M, Proc roy sot (London) B 150 (1959) 53. 15. Muller, H J, Radiation biology (ed A Hollaender) vol. 1, p. 351. McGraw-Hill, New York(1954). 16. Sobels, F H, Repair from genetic radiation damage (ed F H Sobels) p. 179. Pergamon Press, Oxford (1963). 17. Kaufmann, B P, Radiation biology (ed A Hollaender) vol. 1, p. 627. McGraw-Hill, New York (1954). 18. Russell, W L, Russell, L B & Kelly, E M, Science 128 (1958) 1546. 19. - Int j rad biol, suppl. (1960) 311. Exptl Cell Res 69
20. Sobels, F H, Genetical aspects of radlosensitivity Mechanisms of repair. IAEA, Vienna (1966) 49. 21. Traut, H, Genetical aspects of radiosensitivity: Mechanisms of repair. IAEA Vienna (1966) 67. 22. Witkin, E, J cell camp physiol, suppl. 1 58 (1961) 135. 23. Kimball, R F, J cell camp physiol, suppl. 1 58 (1961) 163. 24. Chandley, A C & Kofman-Alfaro, S, Exptl cell res 69 (1971) 45. 25. Evans, E P, Breckon, G & Ford, C E, Cytogenetics 3 (1964) 289. 26. Carr, D H & Walker, J E, Stain technol36 (1961) 233. 27. Kofman-Alfaro, S & Chandley, A C, Chromosoma 31 (1970) 404. 28. Oakberg, E F, Am j anat 99 (1956) 391. 2g. Cleaver, J E, Rad res 30 (1967) 795. 30. Wimber, D E & Prensky, W, Genetics 48 (1963) 1731. 3l. Hotta, Y, Ito, M & Stern, H, Proc natl acad sci US 56 (1966) 1184. 32. Clark, A J & Marguilies, A N, Proc natl acad sci US 53 (1965) 451. 33. Howard-Flanders, P & Theriot, L, Genetics 53 (1966) 1137. 34. Howard-Flanders, P & Boyce, R P, Genetics 50 (1964) 256. 35. Watson, W A F, Mutation res 8 (1969) 91. 36. Riley, R & Miller, T E, Mutation res 3 (1966) 355. 37. Swietlinska, Z & Evans. H 3, Mutation res 10’ (1970) 185.’ 37a. Pearson. P L, Ellis, J D &Evans, H J,,.-Cvtogenetics 9 (19iO) 460. ’ 38. Evans, H J, Brookhaven symp biol 20 (1967) 111. 39. Kihlman, B A & Hartley, B, Hereditas 59 (1968) 439. 40. Muller, H J, J genet 40 (1940) 1. 41. Kaufmann, B P, Proc natl acad sci US 27 (1941) 18. 42. Failla, P M, Science 138 (1962) 1341. Oster, I I, Genetics 40 (1955) 692. 2 Hotta, Y & Stern, H, Biophys biochem cytol 11 (1961j311. 45. Ito, I, Hotta, Y & Stern, H, Dev biol 6 (1967) 54. 46. Whitehouse, H L K, Nature 199 (1963) 1034. 47. Holliday, R, Genet res 5 (1964) 282. 48. Lawrence, C W, Heredity 16 (1961) 83. 49. - Rad botany 1 (1961) 92. 50. - Nature 206 (1965) 789. 51. Lawrence, C w, & Davies, D R, Mut res 4 (1967) 137. 52. havi&, D R & Lawrence, C W, Mut res 4 (1967) 147. 53. Hotta, Y & Stern, H, J mol biol 55 (1971) 337. 54. Stern, H & Hotta, Y, Genetics, suppl. 61 (1969) 55. ZAwell, S H & Stern, H, J mol biol 55 (1971) 357. Received March 26, 1971 Revised version received June 2, 1971
Cell Research 69 (1971) 33-44
TATION-INITIATED DNA SYNTHESIS I CELLS OF THE SUSANA KOFMAN-ALFARO
and ANN @. CMAN
Medical Research Council, Clinical and Population C’ytogenetics Unit, Western General Hospital, Crewe Road, Edinburgh 4, Scotland
SUMMARY 1. “Unscheduled” DNA synthesis has been demonstrated in spermatogenic celis of the mouse following high and low doses of UV and high doses of X-rays, using autoradiographic techniques. 2. A small amount of “natural” DNA synthesis also occurs at the zygotene/pachyteae stages of meiotic prophase. 3. Little or no “unscheduled” DNA synthesis can be detected in testicuiar spermatozoa, but spermatogonia, spermatocytes and spermatids show varying levels of synthesis. 4. Maximum levels of “unscheduled” DNA synthesis are found in late-zygotene/ear?y pachytene spermatocytes, the cell-stage showing some “natural” DNA synthesis in the controls. 5. The results are discussed in terms of a general “repair” system operating in spermatogenic
The detection, by autoradiographic means, of a low level of DNA synthesis following UV irradiation of HeEa cells in all stages of the cell cycle, was first made by Rasmussen & Fainter [I]. This radiation-stimulated synthesis has been termed “unscheduled synthesis” [I!] and has since been found in a variety of other mammalian cell types following both UV and X-n-radiation [3,4]. Caesium chloride density centrifugation studies have shown that the stimulated incorporation of labelled precursor into 6)NA following irradiation of mammalian cells shows features in common with ‘“repair rephcation” observed in bacteria [S, 41. Repair replication occurs at random points along the DNA chain, is non-semiconservative, and in bacteria, is part of a repair process (excision repair), that results in enhanced survival [7]. It is believed that “unscheduled synthesis” and “repair replication” are manifestations 3 - 711818
of the same basic “repair” process HeLa cells, “unscheduled syn been observed after low doses of tion that result in high survival, and it is thus thought that nearly all cells in such populations perform repair replication an retain their reproductive integrity [3]. That the “repaired” DNA of human cells can subsequently undergo normal replication has been shown by Rasmussen et al. [9] and Painter et al. It is well k n that individual stages of oogenesis and spermatogenesis in a variety of organisms vary widely in their se~s~t~v~t~es to irradiation and other agents in terms of cell-killing [I l-141, mutation induction [iZ, 15, 161, and chromosome breakage [97], and while the final radiosensitiv germ-cell stage appears to a variety of factors, the differential capacity of each stage to repair damaged DNA is Exptl Ceil Res 69
34 SusanaKofman-Alfaro & Ann C. Chandley considered to be one of prime importance [16, 18, 19, 20, 211. Evidence from bacteria [22] and Paramecium [23] has shown that repair of pre-mutational damage is invariably associated with DNA synthesis, and we considered it of interest to look for direct evidence of DNA synthesis following irradiation of mammalian germ cells. The present paper gives information from the mouse; our findings for human germ cells will be the subject of a subsequent publication [24]. METHODS The male mice used in the experiments were from a random-bred Q strain and were 6-8 weeks old at the time of killing. Irradiation and 3H-thymidine labelling were carried out in vitro on cellular suspensions of testicular cells prepared by two different methods: (1) For UV irradiations, testes were chopped with fine scissors in sterile saline, until a suspension of germ cells was obtained. The suspension contained all spermatogenic stages from spermatogonia to testicular spermatozoa. In the first series of UV experiments, 1 ml of suspension was irradiated with 2 537 8, light from a low pressure mercury lamp (‘Hanovia’ type) at a doserate of 1 400 ergs/mm2/min in a plastic Petri dish of 3.5 cm diameter. A total dose of 4 200 ergs/mm2 was given. An equal part of suspension formed the unirradiated control. In a subsequent series of experiments, suspensions were irradiated at a dose-rate of 4.2 ergs/nm?/sec to give total doeses of 50 and 100 ergs/mm2. Unirradiated control suspensions were also prepared. Immediately after irradiation, both treated and control suspensions were centrifuged at 1 500 rpm for 5 min, the cells resuspended in 2 ml Eagle MEM (supplemented with 20 % calf serum, 200 IU/ml penicillin and 200 ,ug/ml streptomycin) containing 10 @Zi/ml 3H-thymidine (spec. act. 22 Ci/mmole), and incubated for 2 h at 37°C in a gas phase of 5 % CO, and 95 % air. After incubation, the suspensions were again centrifuged, the supernatant discarded, and the cells resuspended in hypotonic sodium citrate (1 X) for 15 min prior to fixation in 3 : 1 methanol : acetic acid. They were then processed according to the method of Evans et al. 1251and air-dried preparations made. Two replicate runs of the experiment-were performed at the high dose of UV i.e. 4 200 ergs (Runs R2 and R7), and one run at each of the low doses i.e. 50 and 100 ergs (Runs R8 and R9 respectively). (2) For the X-ray experiments, half of each of the right and left testes were exposed to a dose of 5 000 rad in sterile saline, while the other halves were kept as unirradiated controls. Both treated and control Exptl
Cell Res 69
pieces were then incubated for 2 h at 37°C in 5 ml Eagle MEM containing 15 ,&i/ml 3H-thymidine (spec. act. 22 Ci/mmole). After washing in nonradioactive medium, they were transferred to hypotonic sodium citrate (1 %) for 15 min, and then chopped with fine scissors until a suspension of cells was obtained. Air-dried preparations were then made as above [25].
Autoradiography All slides were stained with carbol fuchsin [26] and filmed for autoradioarauhv bv dinning in Ilford L4 liquid emulsion. Adequate au&radiographs were obtained following UV irradiation after a 2 week exposure period. A period of 6 weeks was necessary for cells irradiated with X-rays. Additional slides from each experiment were exposed for 34 months. Some slides from each exneriment were treated with DNAase for 1 h at 37°C in 0.1 M Tris-HCl (pH 7.0) with 0.2 M MeCL which contained 10 r&ml DNAase. Controls were incubated under the same conditions in buffer solution alone. Slides were then washed in distilled water, extracted in 1 % perchloric acid at 4°C for 20 min and washed again.
Definition of cell stages In the testicular suspensions obtained, all stages from spermatogonia to spermatozoa could be recognised. The identification of individual stages was based on our own previous studies of meiosis in the male mouse [27] and on descriptions given by Oakberg
1281.
Treatment of cells by the two different techniques produced slight differences in the final distribution of cells in suspension. For example, fewer spermatogonia, resting primary spermatocytes and leptotene nuclei were seen in suspensions prepared by Method 2 than in those prepared by Method 1. Cells in diplotene were rare in suspensions prepared by Method 1 and absent in those prepared by Method 2. Cells in diakinesis were extremely rare in both.
RESULTS High Dose UV and X-ray Experiments
Unscheduledsynthesis in spermatogoniaand resting primary spermatocytes In control cultures of both the UV and X-ray experiments, heavily-labelled spermatogonia and resting primary spermatocytes, which were in S phase at the time of label incorporation, were found (table 1). These showed various replication patterns depending on the time in the S period at which incorporation had occurred [27].
Unscheduled DNA synthesis in mouse gem cells
35
Fig. 1. Autoradiograph of testicular cells of the mouse following irradiation with a UY dose of 4 200 ergs/mm” and incubation in medium containing 10 &i/ml 3H-thymidine for 2 h. Heavily labelled S period spermatogonia contrast with lightly-labelled late zygotene/early pachytene spermatocytes undergoing unscheduled DNA synthesis. (a) Mid-S pattern (b) end-of-S pattern.
After irradiation with 4 200 ergs/mm2 or 5 000 rad X-rays, two distinct populations of labelled spermatogonia and resting primary spermatocytes were seen. Approximately half the labelled cells were heavily-labelled and were interpreted as S phase cells while the other half were lightly-labelled, and were interpreted as non-S-phase cells showing ‘“unscheduled” DNA synthesis (table 1, fig. 11. ean gram counts per nucleus (based on labelled cells only) for lightly-labelled cells in the two runs of the UV experiment were 2S.Ori: 1.5 and 33.313.1 respectively and the mean for the X-ray experiment was 25.3 i 3.0. Heavily labelled S phase cells in control cultures showed higher levels of incorporation of label than those seen in irradiated cultures. This was particularly obvious following UV irradiation, and it was concluded that some inhibition of S period DNA synthesis had
Table 1. S period and radiation ir?duced “unscheduled” DNA synthesis in spevmatogonia and resting primary spermatocytes following high doses of UV and X4~adiation
No. of cells Run
Control
UV 4 200 ergs R2 100 R7
X-Ray 5000 rad
100
5Q
Treated
No. labelled Cortrol Treated ___ 3% 3% 2 ‘2 3 ‘2
100
1
100
0
100
2
45 62
12
47 40
4i
34
24
31
Low = lightly-labeiled cells performing unschedu?ed DNA synthesis. High = heavily-labelled cells performing S period DNA synthesis.
36
Susana Kofman-Alfaro
& Ann C. Chandley
Table 2. Grain count estimates for various meiotic stages following 4 200 ergs UV irradiation and in controls No. of cells
% labelled
Cell stage
Control
Treated
Control
Treated
Control
Treated
Leptotene a b Zygotene a b Late zygotene/ early pachytene a b Late pachytene a b Diplotene a b 2” Spermatocyte a b Round spermatids a b Elongating spermatids a b Spermatozoa a b
150
200 -1 300 -
2.0 2.7 0.7 9.6
34.0
1.2kO.3 3.6kO.4 1.3kO.2 5.3 kO.4
8.0f1.3
300
6.7 49.0
Autoradiographic 1 Not scored.
75 300 250 300 300 300 250
GO
cpm
53.3 98.5 87.7
1::; 2:::
GO -.
i:
30
200 150 200 150 200 200 200 200
200
0.5
71.0
100
9.0
91.0
500 150 300 300 300 500
0 5.5 0 1.5 0 0
46.6 82.0 2.7 30.0 i.4
3.3 +0.4 13.720.6 2.OkO.3 6.6kO.4
17.3 +2.1 71.213.5 46.8 +3.4
2.4kO.8 7.8kO.9
41.7T8.7 -
0.9F0.2 2.8kO.2 0.5kO.l 2.7i0.2 0.2110.07 0.77+0.09 0.1010.06 0.28iO.05
13.7k1.6 59.Ok3.7 6.2kO.5 40.0i2.6 0.88kO.24 4.51kO.31 0.14+0.04 1.43 10.09
exposure period: (a) 2 weeks (b) 34 months.
occurred following UV irradiation, a phenomenon which has been extensively reported in previous publications [l, 291. The high level of S period labelling in both control and treated groups precluded grain count estimates from being made. Treatment with DNAase resulted in an almost complete disappearance of grains from all cells, showing that label was actually in DNA.
cells were considered to be those having significantly higher counts than the background count for an area of equal size on the slide. In control cultures, after two weeks exposure to the autoradiographic film, a small non-significant increase in incorporation of 3H-thymidine was found in late zygotene/early pachytene spermatocytes relative to other stages (table 2, fig. 2). In slides exposed for 34 months, this peak of Unscheduled synthesis in spermatocytes, labelling was more apparent, the mean count spermatids and spermatozoa per cell at this time being 13.7+ 0.6 (table 2, Grain count estimates for primary and fig. 2). A t test showed this to be signifisecondary spermatocytes, spermatids and cantly higher than the pooled means for spermatozoa following a high dose of UV zygotene (5.3kO.4) and late pachytene irradiation and in their controls are given in (6.6rt: 0.4) (t = 11.27) (P=O.OOl). table 2 and fig. 2. Patterns of labelling are This small peak of “natural” DNA synthesis in the controls corresponds, we believe, shown in fig. 3. Mean counts per cell were based on the to the small amount of prophase DNA total cells scored for each individual stage synthesis which has been claimed to occur i.e. labelled and unlabelled cells. “Labelled” in spermatocytes of the newt [30] and has Exptl Cell Res 69
own biochemically, to occur in 89 rocytes of kilium and Trillium at these stages [31]. (In a recent publication 1231 we reported our failure to detect prophase NA synthesis in male mice injected with TI-thymidine, and concluded this was due to the poor resolution afforded by the auto60 radiographic technique. We now believe that it was due to the fact that insufficient radioactive isotope wa aken up by prophase cells in the testis. th the present in vitro technique, cells have greater opportunity to incorporate label and detection of even low NA synthesis becomes possible.) exposed to a dose of 4 200 -tbymidine incorporation was detected in all spermatogenic stages with the exception of late spermatids and spermatozoa. No incorporation of label was observed in Sertoli cells. Since the meiotic S period in the mouse occurs at preleptotene in the resting primary spermatocyte this incorporation cannot be attributed to normal NA synthesis, and is thus a radiation-stimulated “‘unscheduled” synthesis comL 2 LZiEP Li= D parable to that found in irradiated somatic cells. Significant depressions in grain count were seen after treatment with DNAase, Fig. 2. Abscissa: spermatogenic stage; ordinate: mean 2 weeks autora?iographic grain count/cell. -, showing that the label was in fact in DNA. exposure. ---, 34 months exposure. Among primary spermatocytes, almost Grain count estimates for individual germ-cell stages following irradiation, with a UV dose-of 4 200 10 % of cells in late zygotene/early pachytene ergs/mm2 (T) and in controls (C). L, leptotene; Z, showed an incorporation of 3H-thymidine zygotene; LZIEP, Iate zygotenejearly pachytene; Lk, late pachytene; D, diplotene; 2” Cyte, secondary which far exceeded that of any other prophase spermatocyte; R. tid, round spermatid; E. tid, elongatstage (table 2, fig. 2)~ The mean count per ing spermatid; SF, Spermatozoa. ceb for late zygotene spermatocytes (71.2 k few cells in diakinesis/metaphase I were seen 3.5) was nearly twice that of the next most to make scoring of this stage feasible, of heavily-labelied stage i.e. late pachytene those that were seen, a few (4 out of 16) (40.8 + 3.4), and nearly ten times higher than were labelled with counts ranging from IO-30 the mean for leptotene (S.Oi: 1.3). There was no significant difference in the grains, while the others were un‘iabelied. The mean grain counts for secondary mean grain counts for late pachytene and spermatocytes and round spermatids were diplotene. The large standard error for and 6.22 .5 respectively, thus diplotene was presumed to be due to the 1X7&1.6 showing roughly equal levels of unscheduled low numbers of cells scored. Although too
38 SusanaKofman-Alfaro & Ann C. Chandley
Fig. 3. Unscheduled DNA synthesis in various stages following irradiation. All cells shown are from UV-treated cultures (4 200 ergs/mmz) exposed to film for 2 weeks with the exception of mid-pachytene spermatocytes shown in fig. 3~. These were from X-rayed cultures (5 000 rad) exposed to film for 6 weeks. (a) Zygotene; (b) late zygotene/early pachytene; (c) mid-pachytene; (d) late pachytene; (e) diplotene; (f) diakinesis; (g) 2” spermatocyte; (Jr) round spermatids. Exptl
Cell Res 69
Fig. 4. Autoradiograph of unirradiated control cells showing a low level of “natural” DNA synthesis at the late zygotene/early pachytene stage of meiotic prophase. Autoradiographic exposure period 6 weeks.
iW content, whereas the values for elongating spermatids and spermatozoa were 9.88 +_9.24 and 9.14 + 0.94 respectively. In UV-treated cultures exposed to film for 3$ months, primary spermatocytes were so heavily labelled that accurate grain counting was not possible. The levels of unscheduied DNA synthesis in elongating spermatids and sperm were stiff extremely low, the mean counts for these stages being4.51 k 0.31 and 1.43 i- 9.09 respectively (table 2, fig. 2). In. view of the condition of exposure, it seemed highly unlikely that the absence of significant Iabelling in elongating spermatids and spermatozoa exposed to UV was due to failure of UV penetration. However, in order to clarify this point, the cells were exposed to 5 000 rad X-rays.
The results for a single run of t experiment are given in table 3 and fig. 5. After an exposure period of 6 weeks the control data again showed a small but significant peak of “‘natural” DNA synthesis in late zygotene/early pachytene spermatocytes, the mean count being 5.419.4 grains per cell compared with counts of 2.25 9.2 and 2.0 2 0.2 for zygotene and mid-pachytene respectively and 0.8 5 0.2 for teptotene. After 34 months exposure (table 3), the mean count per cell for late zygotenelearly pachytene was 49.3 F9.9 which was significantly higher than the pooled means for zygotene (3.7 * 0.5) and mid-pachytene (3.4 I 0.5) (t=6.75; P= .OOZ). The results for the controls of both the UV and X-ray experiments thus indicate that natura’f DNA
40
Susana Kofman-Alfaro
h Ann C. Chandley
Table 3. Unscheduled DNA synthesis following 5 000 rad X-rays No of cells
% labelled
Stage
Control
Treated
Control
Leptotene a
41 50 200 100
100 -1 150
0 2.0 3.5 8.0
300 100 150 100 150 50
150 150 100 -
16.0 29.0 2.0 6.0 0 0
150 100 150 100 100 100 100 100
300 100 300 100 200 200 200 200
0 3.0 0 1.0 0 0 0 0
b Zygotene a b
Late Zygotenej Early pachytene a b
Mid pachytene a b Late pachytene a b
2” Spermatocyte a b
Round spermatid a b
Elongating spermatid a b
Spermatozoa a b
Autoradiographic l Not scored.
Cell Res 69
Treated 3.0
47.3 73.3 ii.0 -
8.0
18.3 25.0 19.7 42.0 0.5 8.0 0 0.5
Control
Treated
0.8 io.2 1.6iO.4 2.210.2 3.7kO.5
1.3 +0.4 14.321.3 -
5.420.4 10.3 LO.9 2.oio.2 3.6kO.5 1.0~0.1 1.9kO.4
37.7 k2.5
0.6 LO.1 1.220.2 0.5 +0.1 1.7iO.2 0.24 kO.05 0.37 kO.08 0.12+0.03 0.21 kO.05
9.6T1.3 4.5i1.3 3.3 jo.3 7.151.1 3.3 +0.3 lO.Oi1.3 0.15 io.05 1.56f0.21 0.07 10.02 0.40 10.09
exposure period: (a) 6 weeks; (b) 39 months.
synthesis occurs throughout the zygotene and pachytene stages of prophase reaching a peak at late zygotene/early pachytene. No significant prophase synthesis occurs at leptotene. The results for the X-irradiated cultures after an exposure period of 6 weeks confirmed our main findings with UV, showing (i) late zygotenelearly pachytene was the stage at which maximum levels of 3H-thymidine incorporation occurred, and (ii) elongating spermatids and spermatozoa showed little, if any, incorporation (table 3, fig. 5). (iii) No incorporation was seen in Sertoli cells. The mean grain count for late zygotene/early pachytene after 6 weeks’ exposure of the films, was 37.7k2.5 (approximately half that obtained with UV after only 2 weeks’ exposure). After 3& months exposure primary spermatocytes in the X-rayed cultures showed levels of incorporation which were too high for accurate grain estimates to be made. As in Exptl
cpm
the UV experiments elongating spermatids and spermatozoa showed negligible incorporation (table 3, fig. 5). Low Dose UV Experiments
The UV dose of 4 200 ergs/mm2used in the first experiments was almost certainly lethal to the cells exposed. The question arises therefore, whether the labelling observed is part of a pathological process, leading to cell death. Clarkson & Evans (personal communication) in our laboratory have shown that a maximum incorporation of label occurs in human lymphocyte DNA after exposure to 40 ergs. Mouse germ cells therefore were exposed to doses of 50 and 100 ergs/mm2. Selected stages at each level of ‘ploidy’ were scored and the results are given in table 4 and fig. 6. The results show that unscheduled synthesis is clearly evident following low dose exposures.
Table 4. Unscheduled
NA synthesis in ceh exposed to UT/ doses QJ”50 and i&3 ergslmna2 -Dose No. % Stage
mm2
Zygotene
50 100 Late zygotene/ 50 Early pachytene 100 2” Soermato&e Round spermatids
RXd
ETid
Labelled
cpm
-
50 50 50
44.0 42.0 100.0
9.9k1.5 14.1 k2.5 46.8 -c 3.0
50
96.0
54.5*4,1
50 IO0
5 50
58.0 66.0
7.9 jo.9 14.1 xl.7
50 100
100 50
64.0 58.0
Autoradiographic
‘Cyte
Cells
6.8 iO.6 8.6&1.1
exposure period 2 weeks.
Sp
4c------l~zc+r----cDNA CONTENT
Fig. 5. Abs&8ssa: spermatogenic stage; ordinate: mean grain count/cell. -, 6 weeks autoradiographic exposure; ---, 34 months exposure. Grain count estimates for individual germ-cell stages following irradiation with 5 000 rad X-rays (T) and in controls (C). Abbreviations as in fig. 2. MP, mid-pachytene.
Localization of DNA Synthesis
The possibility existed that some of the NA we were observing could have been in the cytoplasm closely surrounding the nucleus and not in the nucleus itself. That this was not so, was evident from slides stained with the fluorescent stains acridine orange and “Atebrin”, which showed that most of the nuclei in the preparations were naked and free of cytoplasm. Cytoplasmic Pabelling was however found around many human spermatogenic cells [24] and when these were stained with “Atebrin” it was clear that the cytoplasm still adhered to most of the nnciei. Why cytoplasm should be shed from mouse cells but not from human cells, when the technique used in both cases is the same, is unknown.
Z w DNA CONTENT
LZiEP 4c~-Pc+------C--------d
-cyte
RTid
Fig. 6. Grain count estimates for selected germ-cell stages following irradiation with high and low doses of UV, -, 4 200 ergs/mm2; ---, 100 ergs/mm2; -.-.-., 50 ergs/mm2. Exposure to autoradiographic film 2 weeks. Abbreviations as in figs 2 and 5.
42
Susana Kofman-Alfaro
& Ann C. Chandley
DISCUSSION We have demonstrated that “unscheduled” DNA synthesis is initiated in spermatogenic cells of the mouse following exposure to UV or X-rays and that the level of stimulated synthesis varies from one germ-cell stage to another. Furthermore we have found a similar pattern of response in germ cells subjected to “high” and “low” doses of UV and to “high” doses of X-rays, suggesting that the same basic process may be operating at all dose levels and with both kinds of radiation. Whether this stimulated synthesis is part of a repair process that restores damaged DNA to its original state is not clear. In its broadest sense, the term “repair” applied to irradiated meiotic cells could be considered in terms of enhanced survival, reduced mutation frequency (“repair of premutational damage”) and should also include events resulting in chromosome exchange and possibly the normal process of exchange, associated with crossing-over. If “repair” of DNA is involved in all these phenomena, there is no evidence that this “repair” is the same for all these events or even that common mechanisms are involved. However, evidence from bacteria has revealed an association between recombination and repair of UV-induced damage. Mutants of E. coli K12 have been isolated which are unable to form recombinants by conjugation (Ret-) and are also unable to repair photodamaged DNA [32, 331 and it has been suggestedthat certain enzymes and common pathways may serve both in genetic recombination and in repair following irradiation [34]. In eukaryotes there is also some evidence that recombination-deficient mutants may be radiation-sensitive. Watson [35] has shown, for example, that the recombination-deficient c3G of Drosophila melanogaster is more radiation-sensitive than wild-type flies in Exptl
Cell Res 69
terms of dominant lethal induction. Also the data of Riley & Miller [36] suggeststhat a desynaptic mutant in barley is more radiation-sensitive in terms of aberration yield than the wild type, although confirmation of this could not be obtained in more extensive experiments carried out by Swietlinska & Evans [37]. Finally in man, a patient showing a desynaptic condition in most spermatocytes at diakinesis has been found to show reduced levels of “unscheduled synthesis” in the blood [37a]. It has been suggested 138, 391 that the mechanisms involved in the exchange which occurs at meiosis to give chiasmata and crossing-over may be the same as those leading to the formation of chromosome aberrations. If our unscheduled synthesis is indeed part of a “repair” process operating at meiosis, some interesting correlations with the genetic data on repair in germ-cells can be seen.It is known from work on Drosophila [40, 411 the mouse [18, 191 and sea urchin [42] that sperm show no repair, following irradiation, until fertilization. Other gametogenie stages, e.g. spermatogonia, spermatids and oocytes, are assumed to undergo some repair on the basis of evidence from doserate and other experiments [18, 19,431. If our unscheduled DNA synthesis in the mouse is a manifestation of “repair”, then our results would support the genetic finding of “no repair in sperm” by showing little or no unscheduled synthesis in them. The lack of repair in sperm may be due to their low metabolic activity [18, 211 or may be a consequence of the fact that the DNA in sperm is tightly packaged in basic protein and may not be accessibleto repair enzymes. If the “repair capacity” of different germcell stagesis associated with intrinsic factors such as metabolic activity, the finding of maximum levels of unscheduled DNA synthe-
sis in late zygotene/early pachytene spermatocytes may be associated with a high metabolic activity. The enormous growth of spermatocytes from leptotene to pachytene suggestsincreasing metabolic activity. One of the most striking findings in our experiments is a highly significant peak of -thymidine incorporation at late zygotene/ early pachytene in irradiated cells and a coincident peak of “‘natural” DNA synthesis in the controls. “‘Natural” prophase DNA synthesis has also been found to occur in microsporocytes of Lilium and Trillium at the zygotenej pachytene stages[31,44,53] and this synthesis has recently been analysed by the techniques Of A-DNA hybridisation, density-labellin nd by the use of the DNA synthesis inhibitor hydroxyurea [53]. Zygotene synthesis in Eilium appears to represent a delayed semiconservative replication of approx. 0.3 Y0 NA and shows a higher G + C lk nuclear DNA. Its function in meiosis appears to be associated with chromosome pairing [64]. DNA synthesised during pachytene represents approx. 0.1% of the genome and appears to have the characteristics of a “repair replication” [53]. The ilure of bromodeoxyuridine-substituted NA synthesisedduring pachytene to show a “‘heavy shift” in buoyant density and the ineffectiveness of hydroxyurea in arresting pachytene DNA synthesis are taken as evidence to support this conclusion. Pachytene NA synthesis is believed to be associated with the exchange process resulting in crossing-over c45, 531. Although there is no direct evidence that prophase synthesis of DNA is involved in crossing-over its occurrence has come to be associated with those hypotheses which postulate a “repair’‘-type NA as the terminal event in the crossing-over process [46, 471. Indirect evidence has now accumulated which suggests
that a critical step in the crossing-over process does occur at the zygotenejpac meiotic prophase [48, 49, 501 step involves the synthesis of Furthermore, owe11 6% Stern [55] have recently identified a set of ~Qrn~Ie~~~tary d repair enzyme activities
believe may sponsor a breakage-reunion mechanism responsible for genetic recombiur findings of coinci NA synthesis a initiated” “ unscheduled” synthesis in mouse spermatocytes at late zygotenelearly pachytene correlate well with the increased endonuclease, polynucleotide kinase and ligase activities found during this stage in kllilam [55], and if ow unscheduled in the mouse represents t damaged NA our results may indicate common enzymic activities in the phenomena of breakage, excision and exchange in recombination and in t e repair of UVdamaged DNA. If at least a part of the ““natural” synthesis occurring at prophase in the is a semi-conservative replication similar to that occurring during the S period, we might have expected inhibition of this synthesis following irradiation. Inhibition of NA synthesisfollowing irradiation has been uE.d in S phase cells of the testis and in S phase somatic cells [l, 3, LB]. On the contrary, our results showed an enormous increase in incorporation of label into the DNA of late zygotenelearly pachytene spermatocytes-the stage at which “natural” DNA synthesis was found. This amounted to an g-fold increase in X-rayed cultures and a 25-fold increase in UV-treated cultures as compared with controls. The amount of stimulated synthesis observed at this time is als greater than the unscheduled
44 SusanaKofman-Alfaro & Ann C. Chandley induced in cells in other stages of meiosis at the time of irradiation. It seemsprobable therefore that the large amount of radiation induced synthesis at zygotene/pachytene may reflect the involvement of a recombinational repair process that is normally operative, at a much reduced level in ‘control’ cells at this time. Unscheduled synthesis at the other meiotic stages may be more comparable to that observed in somatic cells. The authors are grateful to Professor H. J. Evans for his help and advice during the course of this work, and for his criticism of the manuscript. They also wish to thank Mrs Judy Fletcher for excellent technical assistance and Mr Norman Davidson for his help in the preparation of the photographs.
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