Studies on mutagen-sensitive strains of Drosophila melanogaster

Mutation Research, 149 (1985) 415-419 Elsevier

415

MTR 04036

Studies on mutagen-sensitive strains of

Drosophila melanogaster

VII. Effects of repair deficiency in males on X-ray-induced sex-linked recessive lethals in spermatozoa K. Sankaranarayananand W. Ferro Department of Radiation Genetics and Chemical Mutagenesis, Syloius Laboratories, State University of Leiden, Wassenaarseweg 72, 2300 RA Leiden (The Netherlands) and The J.A. Cohen Interuniversity Institute for Radiopathology and Radiation Protection, Leiden (The Netherlands) (Received 10 September 1984) (Revision received 20 December 1984) (Accepted 21 December 1984)

Summary The response of mature spermatozoa to the X-ray induction (500 R and 3000 R) of sex-linked recessive lethals was studied in Drosophila melanogaster males known to be deficient in excision- or post-replication repair of UV damage in somatic ceils. The results show that the induced frequencies of recessive lethals in the excision-repair-deficient males (mei-9 a and mei-9 L1) are similar to those in the appropriate repair-proficient males (mei ÷ and Berlin-K). However, in the post-replication-repair-deficient males (w mus(1)101 ol), these frequencies are significantly lower than in the comparable repair-proficient males (w) after 500 R, but not after 3000 R.

Muller demonstrated, already in 1940, that chromosome breaks induced in mature spermatozoa do not rejoin before fertilization, the inference being that chromosomal damage induced in this germ cell stage is not amenable to repair by paternal repair processes. Sobels (1974) showed that this is also true of breaks induced in late spermatids (sampled from 36-38-h-old male pupae) whereas those induced in early spermatids (sampled from 24-h-old pupae) undergo repair before fertilization. It thus appears that the paternal repair system is 'switched off' after the early spermatid stage in spermiogenesis. The findings of Wi~rgler and Maier (1972) and Wiargler et al. (1972) that the yields of sex-chromosome losses induced in spermatozoa can be modified by using females of different genotypes strongly suggested

that the matemal repair processes play an important role in the realization of chromosomal damage induced in this cell stage. This 'maternal effect' approach has been successfully exploited over the years, and more recently, using mutant strains known to be deficient in specific DNA-repair pathways, to gain some insights into the genetic control of DNA repair in Drosophila germ cells. Some of the maternal effect studies using repair-deficient mutants have been recently reviewed by Sobels et al. (1984). The problem of whether and to what extent, 'mutational damage' induced in male germ cell stages is subject to the action of paternal repair processes, and whether strains deficient in DNArepair pathways would show altered responses relative to repair-proficient strains has, until recently,

0027-5107/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

416

attracted less attention. In large-scale experiments carried out in the 1960s, Sobels (1964, 1965, 1966) showed, among other things, that when males were X-irradiated under anoxia and then post-treated with either N 2 o r O2, the yields of both sex-linked recessive lethals and autosomal translocations were significantly lower with N 2 post-treatment in spermatozoa, but after 02 post-treatment in early spermatids. The question of whether these posttreatment effects, particularly the one observed in spermatozoa is a consequence of repair of potential lesions (leading to mutations or chromosome aberrations) in the cell stage itself (and this was the interpretation advanced by Sobels at that time) or whether the combination of treatments to which these germ cells were exposed made them more, or less, susceptible to the repair processes in females (see Sankaranarayanan and Sobels, 1976) has not yet been satisfactorily answered. In more recent studies, Smith et al. (1983) showed that when males of the repair-deficient strain mei-9 Avl (and of a repair-proficient control strain) were treated with the alkylating agents ethyl methanesulphonate and methyl methanesulphonate, the yields of sex-linked recessive lethals were 4-8 times higher in spermatogonia of the rnei-9 AT1 strain; however, the responses of meiotic and post-meiotic cell stages were similar in both strains. The authors suggest that an excision-repair process functions until the commencement of meiosis and that the enhanced sensitivity of the spermatogonia of the repair-deficient strain is most likely a consequence of the defect in excision-repair of damage produced by the alkylating agents. In a comparative study of the responses of early spermatids and spermatogonia of mei-9 a, mei-41 D5 and mus(1)lO1 DI males to X-irradiation, Eeken and Sobels (1985) demonstrated that these germ cells responded with higher frequencies of sex-linked recessive lethals only in mei-9 ~ males and that the effect was more pronounced in spermatogonia. In a complementary study, we explored further the question of whether paternal repair processes have ceased functioning in mature spermatozoa, by comparing the response of this germ cell stage to the X-ray induction of sex-linked recessive lethals in males of three repair-deficient (mei-9 a, mei-9 La and w mus(1)lO1D1) and in those of appropriate repair-proficient strains. The results are presented in this paper.

Material and methods

Males from the following strains were used: (i) y mei-9a; bw; st p P / y + . Y (deficient in excision

repair of UV-damage in somatic cells, besides being sensitive to the killing effects of UV, X-rays, methyl methanesulphonate etc. in the larval stages; Boyd et al., 1976); (ii) y mei +," bw," st pP spaP°l/ y+- Y (repair-proficient control for (i) above; both these strains have markers on chromosomes II and III and were originally derived to facilitate combined recessive lethal and II-III translocation tests in maternal effect studies; Ferro, 1983); (iii) w m u s ( 1 ) l O l O l / B S . Y (deficient in post-replication repair of UV damage in somatic cells besides being sensitive to the killing effects of UV, X-rays, methyl methanesuiphonate, etc.; Boyd and Setlow, 1976); (iv) w (repair-proficient control for (iii) above); (v) mei-9 L1 (originally derived from Berlin-K," mei-9 L1 is allelic to mei-9a; Graf et al., 1979) and (vi) Berlin-K (repair-proficient control for (v) above). In what follows, the following abbreviations will be used: y mei-9a: mei-9a; y mei+; bw; stpP; spaP°l/y+.Y: mei+; w mus(1)lO1D1/B s. Y: w rnus-101.

Two series of experiments were carried out. In the first, 3-5-day-old mei-9 and mei + males were pre-treated with N2, air or 02 for 30 min and X-irradiated with 3000 R, 1500 R and 1500 R, respectively; there were no post-treatments with gases. The irradiated males were mass-mated to Base (Muller-5) females for 24 h after which the males were discarded and the females subcultured for 2 days. The F 1 females were used for sex-linked recessive lethal assays. In the second series, mus101, w, mei-9 LI and Berlin-K males were X-irradiated (without any gas pre-treatment) with either 500 R or 3000 R and mated to females of a modified Muller-5 stock ( y w a B real). The remainder of the procedure was the same as in series I. In both series, appropriate unirradiated controls were run concurrently. The X-irradiations were administered with an ENRAF machine operated at 105 kV and 12 mA at an exposure rate of about 50 R/sec. All exposures were monitored with a Philips Universal dosimeter or with a PTW Dosimentor system. Statistical comparisons were made by calculating standard normal T-values according to Fleiss

417

marginally higher than that for Berlin-K males (P = 0.045, Fisher's exact test and P = 0.043 based on the sum of T values ( = - 2 . 0 0 7 ) for the 6 individual experiments). However, when the induced frequencies in rnei-9 L1 and Berlin-K males are compared (i.e., 1.69% versus 1.41%), the difference is not significant (P = 0.21). The response of w rnus-101 males to 500 R however, presents a different picture: the observed frequency of 1.22% is significantly lower than 2.16% for w males (P 0.001 in Fisher's exact test on pooled data and P 10 -6 based on the sum of T values ( = 5.070) for the 5 individual experiments). Furthermore, the data from individual experiments are remarkably consistent ( w mus-101: 29/2290 (1.27%); 14/1514 (0.92%); 31/2270 (1.37%); 33/2233 (1.48%); 22/2272 (0.97%); w: 33/2197 (1.50%); 24/1073 (2.24%); 43/1779 (2.41%); 48/2029 (2.37%) and 49/2062 (2.38%). When comparisons are based on induced frequencies (i.e., 0.91% versus 1.69%), the responses of these two strains remain significantly different (P = 0.0002). At the higher exposure of 3000 R however, there are no significant differences in response between repair-deficient and repair-proficient strains (P = 0.48 in the w versus w mus-101 and P = 0.40 in the Berlin-K versus mei-9 L1 comparisons). Thus, the present study shows that (i) excisionrepair deficiency in the male (exemplified by the

(1973) (see also Mendelson, 1974). The results of different experiments were combined to give overall P values (2-sided test) for differences between the groups compared. Results and discussion

The results of the first series of experiments are given in Table 1 and those of the second, in Table 2. Except for the 3000-R experiments in series II, all the others were carried out at least twice. Since there were no significant heterogeneities, the data for each strain at each exposure level were pooled. Inspection of Table 1 will reveal that (i) the spontaneous frequency is significantly higher in mei-9 than in mei ÷ males and (ii) in the radiation experiments, there are no significant differences in response between the mei-9 and the mei ÷ males and this is true irrespective of the gaseous atmosphere used during the irradiations. The data given in Table 2 show that the spontaneous rates are similar in both repair-deficient and repair-proficient males (w mus-101 m versus w, P = 0.18 and rnei-9 L~ versus Berlin-K, P = 0.28; Fisher's exact test on pooled data). It is worth mentioning that the spontaneous rates of 0.31% (w mus-101) and 0.47% (w) are higher than those recorded in the work of Mason (1980; w mus-101: 0.05% and w: 0.15%). After an X-ray exposure of 500 R, the observed frequency in mei-9 L1 males is

TABLE 1 F R E Q U E N C I E S OF S E X - L I N K E D RECESSIVE L E T H A L S IN C O N T R O L A N D I R R A D I A T E D m e i ÷ A N D m e i . 9 ( S A M P L I N G OF S P E R M A T O Z O A ) The P values given are for the hypothesis of no difference between the mutation frequencies in m e i + a n d m e i - 9 non-significant. N u m b e r of Expts.

Radiation exposure (R) and pre-treatment

N u m b e r of Xchr. tested

mei ÷

4 4

-

3 661 3 571

5 22

0.14 (0.04-0.32 0.62 (0.39-0.93

0.003

2 2

3000 in N 2 3000 in N 2

1331 1172

73 63

5.48 (4.32-6.85 5.38 (4.15-6.83

NS

mei-9

3 3

1500 in air 1500 in air

2204 3 404

91 177

4.13 (3.34-5.05) 5.20 (4.48-6.00)

NS

mei ÷

2

1500 in 0 2

1117

86

7.70 (6.20--9.42)

mei- 9

2

1500 in 02

1 152

64

5.60 (4.30-7.04)

mei ÷ mei-9 mei ÷

Frequency (%) and 95% confidence limits

males. NS,

Genotype of males

mei- 9

N u m b e r of lethals

MALES

P

NS

418 TABLE 2 F R E Q U E N C I E S OF S E X - L I N K E D RECESSIVE L E T H A L S IN C O N T R O L A N D I R R A D I A T E D R E P A I R - P R O F I C I E N T A N D R E P A I R - D E F I C I E N T MALES ( S A M P L I N G O F S P E R M A T O Z O A ) The P values given are for the hypothesis of no difference between the mutation frequencies in the repair-proficient and repair-deficient males. NS, non-significant. Genotype of males N u m b e r of Radiation Expts. exposure (R) w

N u m b e r of Xchr. tested

N u m b e r of Frequency (%) and 95% P lethals confidence limits

Induced P frequency (~)

5 5

-

6607 7308

31 23

0.47 (0.33- 0.66) 0.31 (0.20- 0.47)

NS

5 5

500 500

9140 10579

197 129

2.16 (1.87- 2.47) 1.22 (1.02- 1.45)

< 1 0 - 6 1.69 0.91

0.0002

1 1

3000 3 000

1021 1148

77 98

7.61 (6.05- 9.42) 8.54 (6.98-10.30)

NS

6.14 8.23

NS

m u s - 101 Berlin-K m e i - 9 L1

5 5

-

6829 6537

21 27

0.31 (0.19- 0.47) 0.41 (0.27- 0.60)

NS

-

-

Berlin-K m e i - 9 L1

6 6

500 500

12204 12 700

210 264

1.72 (1.50- 1.97) 2.08 (1.84- 2.34)

0.045

1.41 1.67

NS

Berlin-K

1 1

3000 3000

1850 1336

168 109

9.08 (7.81-10.48) 8.16 (6.75- 9.76)

NS

8.77 7.75

NS

mus-101

w mus-101

w

m e i - 9 L1

results with mei-9 a and mei-9 L1 mutants) does not lead to any measurable difference in the response of the mature spermatozoa to the X-ray induction of sex-linked recessive lethals and (ii) post-replication-repair deficiency in the male however, may lead to an altered mutational response in spermatozoa depending on the X-ray exposure (lower yields after 500 R, but not after 3000 R, relative to repair-proficient males). These findings viewed in the context of the work of Ferro and Eeken (1985) with late spermatids and mature spermatozoa (maternal effect studies) and of Eeken and Sobels (1985) with early spermatids and spermatogonia (paternal repair studies) lend credence to the notion that the paternal excision repair process is probably switched off after the early spermatid stage whereas this may not be so in the case of paternal post-replication-repair process, at least at low X-ray exposures. One should hasten to add however that, since there is no DNA replication in the sperm nucleus until after fertilization, it is difficult to envisage a functioning post-replication repair process in spermatozoa. If the lowered recovery of mutations with mus10l males is in fact a consequence of the post-replication-repair deficiency, then one needs the as-

-

-

sumptions that (i) the paternal post-replication-repair process is turned on after fertilization at around the same time when the maternal repair processes become activated in the inseminated oocyte and (ii) that the final outcome is determined by a balance between the operations of both paternal and maternal repair processes. Alternatively (i) it may be that in mature spermatozoa of mus-101 males the chromatin structure is somewhat different from that found in wild-type sperm; some support for this possibility comes from the experiments of Gatti et al. (1983) who found differences in the condensation of chromosomes between mus-101 and wild-type neural ganglia of larvae. At low levels of X-ray-induced damage, this structural difference might make the lesions better accessible to the maternal repair systems and (ii) the differences at 500 R might just be a chance variation; if one plots the data, the w and mus-101 data seem to fit the same linear response. The statistical significance of the 500 R point may therefore be the result of 10-fold higher number of tests (relative to those for 300 R) and of a (really unexpected) systematic difference between the sets in 5 replicate experiments. In view of the uncertainties associated with

419 e x t r a p o l a t i o n f r o m p o s t - r e p l i c a t i o n r e p a i r defects i d e n t i f i e d i n s o m a t i c cells after U V i r r a d i a t i o n to s u c h defects i n g e r m cells after X - i r r a d i a t i o n a n d the v i r t u a l lack of k n o w l e d g e o n the relative i m p o r t a n c e o f p o s t - r e p l i c a t i o n - r e p a i r processes i n the genesis of r a d i a t i o n - i n d u c e d m u t a t i o n s i n D r o sophila, f u r t h e r s p e c u l a t i o n s are p r e m a t u r e at the p r e s e n t time. Acknowledgements W e wish to t h a n k Prof. F . H . Sobels a n d J.C.J. E e k e n for u s e f u l discussions, Prof. F.E. Wi~rgler for his t h o u g h t f u l c o m m e n t s a n d Mrs. A. v a n D u y n , Miss M.J. L o o s a n d Miss I. K l i n k for a b l e t e c h n i c a l assistance. T h e w o r k was i n p a r t supported by Euratoms Contract DI-BI-E-406-81-NL w i t h the U n i v e r s i t y of L e i d e n .

References Boyd, J.B., and R.B. Setlow (1976) Characterization of postreplication repair in mutagen-sensitive strains of Drosophila melanogaster, Genetics, 84, 507-526. Boyd, J.B., M.D. Golino and R.B. Setlow (1976) The mei-9 a mutant of Drosophila melanogaster increases mutagen sensitivity and decreases excision repair, Genetics, 84, 527-544. Eeken, J.C.J., and F,H. Sobeis (1985) Studies on mutagen-sensitive strains of Drosophila melanogaster, VI. The effect of DNA repair deficiencies in spermatids, spermatocytes and spermatogonia irradiated in N 2 or 02, Mutation Res., 149, 409-414. Ferro, W. (1983) Studies on mutagen-sensitive strains of Drosophila melanogaster, II. Detection of qualitative differences between genetic damage induced by X-irradiation of mature spermatozoa in oxygenated and anoxic atmospheres through the use of the repair-deficient mutant mei9 a, Mutation Res., 107, 79-82. Ferro, W., and J.C.J. Eeken (1985) Studies on mutagen-sensitive strains by Drosophila melanogaster, IV. Modification of genetic damage induced by X-irradiation of spermatozoa and spermatids in N 2 or 02 by mei-9 a, mei-41 D5 and mus(l)101 nl, Mutation Res., 149, 385-398. ]~ieiss,~J.L. (1973) Statistical Methods for Rates and Proportions, Wiley, New York. Gatti, M., S. Pimpinelli, C. Bove and S. Bonaccorsi (1983) Genetic control of chromosome breakage and rejoining in Drosophila melanogaster, in: Mechanisms of Production of

Radiation-induced Chromosome Anomalies in Eukaryotes, Euratom Meeting, Brussels (25-26 Oct. 1983), Abstract No. 8. Graf, U., E. Vogel, U.P. Biber and F.E. Wiirgler (1979) Genetic control of mutagen-sensitivity in Drosophila melanogaster, A new allele at the mei-9 locus on the X-chromosome, Mutation Res., 59, 129-133. Mason, J.M. (1980) Spontaneous mutation frequencies in mutagen-sensitivemutants of Drosophila melanogaster, Mutation Res., 72, 323-326. Mendelson, D. (1974) The effect of caffeine on repair systems in oocytes of Drosophila melanogaster, Mutation Res., 22, 145-156. Muller, H.J. (1940) An analysis of the process of structural change in chromosomes of Drosophila, J. Genet., 40, 1-66. Sankaranarayanan, K., and F.H. Sobels (1976) Radiation genetics, in: M. Ashburne and E. Novitski (Eds.), Genetics and Biology of Drosophila, Vol. 1C, Academic Press, New York, pp. 1089-1250. Smith, P.D., C.F. Brumen and R.L. Dusenbery (1983) Mutagen sensitivity of Drosophila melanogaster, VI. Evidence from excision-defective mei-9 ATI mutant for the timing of DNA repair activity during spermatogenesis, Mutation Res., 108, 175-184. Sobels, F.H. (1964) Post-radiation reduction of genetic damage in mature Drosophila sperm by nitrogen, Mutation Res., 1, 472-477. Sobels, F.H. (1965) The role of oxygen in determining initial sensitivity and post-radiation recovery in successive stages of Drosophila spermatogenesis, Mutation Res., 2, 168-191. Sobels, F.H. (1966) Processes underlying repair and radiosensitivity in spermatozoa and spermatids of Drosophila, in: Genetical Aspects of Radiosensitivity: Mechanisms of Repair, International Atomic Energy Agency, Vienna, pp. 49-65. Sobels, F.H. (1974) The persistence of chromosome breaks in different stages of spermatogenesis of Drosophila, Mutation Res., 23, 361-368. Sobeis, F.H., J.C.J., Eeken, W. Ferro, H. Frei, B. Leigh, K. Sankaranarayanan and E.W. Vogel (1984) Studies of mutation process in Drosophila by means of repair-deficient mutants, in: Genetics: New Frontiers, Proc. XVth Int. Cong. Genet., New Delhi, India, December 1983, Oxford and IBH Publishers, New Delhi, pp. 317-328. W~rgler, F.E., and P. Maier (1972) Genetical control of mutation induction in Drosophila melanogaster, I. Sex chromosome loss in X-rayed mature sperm, Mutation Res., 15, 41-53. Wiirgler, F.E., P. Maier and M. Kalin (1972) Maternal effects of sex-chromosome loss in X-rayed mature sperm of Drosophila melanogaster, Arch. Genet., 45, 53-59.