X-RAY INDUCED MUTATIONS IN SPERMATOGONIAL CELLS OF DROSOPHILA AND THEIR DOSE-FREQUENCY RELATIONSHIP1

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1 X-RAY INDUCED MUTATIONS IN SPERMATOGONIAL CELLS OF DROSOPHILA AND THEIR DOSE-FREQUENCY RELATIONSHIP1 S. ABRAHAMSON AND L. D. FRIEDMAN2 Department of Zoology and. Department of Genetics, University of Wisconsin, Madison Received November 2, 1963 INTEREST in the mutability of spermatogonial cells has increased in recent years, primarily due to the demonstration of an X-ray intensity effect on these cells in the mouse (RUSSELL, RUSSELL, and KELLY 1958; RUSSELL 1960). Relatively few mutation experiments in Drosophila have utilized these cells compared to sperm and spermatid cells, and no large scale dose-frequency study has been carried out on spermatogonia. Because of the implications to induced genetic effects on human immature germ cells, it seemed desirable to determine the dosefrequency response of Drosophila spermatogonia to irradiation. These studies developed as an outgrowth from experiments considering the possibility of reparation of X-ray damage (ABRAHAMSON 1961) and because very preliminary data (unpublished) suggested that there might be a falling off of the curve at higher doses indicating the possibility of cell heterogeneity; we therefore sought more information on this subject. This paper presents some of the data discussed in an earlier preliminary communication ( ABRAHAMSON and FRIEDMAN 1962). MATERIALS AND METHODS Wild-type males of the Canton-S strain were collected and irradiated at 24 to 72 hours of age. These males were mass-mated with excesses of virgin females (at least two to three times as many) in quarter-pint milk bottles for 15 days when, as other studies have indicated, cells which were spermatogonia at the time of treatment would be sampled. Every two to three days during this period extra females collected as automatic virgins by the prune killer techniques were added to the new cultures to which the males were transferred. After every six-day period the flies were etherized, the females were discarded and fresh virgin females were added to prevent the cultures from becoming too crowded. Thus in the later broods because of the death of many males there were from ten to twenty times as many females as males per culture. At the end of the brooding period the males were singly mated with two or three virgin females. The female stock in one of the series was the scs1b ZnS sc8 apr/x.y and in other experiments homozygous scslb ZnS sc8apr (Basc or Muller-5) females were used. The former stock was used to simplify virgin collecting since, by the procedure of preparation, the brothers of these females were sterile for lack of a Y chromosome (except if nondisjunction occurred). In the first set of experiments described, a single F, heterozygous Bar-eyed female was collected from each F, culture and mated with several Basc males obtained from stock cultures. This method eliminated the problem of Contribution 926 from the Department of Genetics. This research was supported in part by grants from the National Science Foundation (G193M) and the Wisconsin Alumni Research Foundation, and in part by a grant from the National Institutes of Health (RG 7666). Present address: Department of Biology, Hiram College, Hiram, Ohio. Genetics 49: FebruaIy 1964.

2 358 S. ABRAHAMSON AND L. D. FRIEDMAN clustering, resulting when a mutation arises in a primitive germ cell, which subsequently produces a large number of identically mutant descendant cells. It was also decided that it would be of interest to determine the extensiveness of clustering of lethals. Therefore, a second small scale experiment was carried out, wherein from each male ten F, daughters (if obtainable) were singly mated. In all experiments the F, cultures were scored for sex-linked recessive lethal mutations. If less than 10 percent of the expected number of males were wild type and at least 20 Basc males appeared, the culture was classified as lethal. Confirmatory tests were carried out on all lethals and suspected lethal cultures, using the same criterion of lethality. Thus, two generations were examined before a final classification was established. Many of the lethals have been maintained for further analysis. The irradiations were all performed using a GE Maxitron X-ray machine with 1 mm aluminum filter, operating at 30 ma and 250 KVP with a dose rate of 585 r/min at 37 cm target distance. We are indebted to DR. H. VERMUND and his colleagues of the Department of Radiology for permitting us the use of this machine. The dose was measured by a Victoreen dosimeter and was continuously monitored by a Victoreen iometer which indicated that the dose rate was always within plus or minus 2 percent of the measured rate. Moreover, in one experiment we kept lithium fluoride capsules alongside the flies during the irradiation. This chemical dosimetry was in excellent agreement with the standard physical dosimetry. MR. G. KENNY of the Department of Radiology performed the chemical dosimetry analysis for us. In order to cut down sterility induced by the higher X-ray doses, the X rays were administered in two equal fractions separated by a 24-hour interval for all irradiation doses, which included 3,000, 9,000, and 12,OOOr in the noncluster experiment and 600, 3,000, and 6,000r in the cluster experiment. Since it was not feasible in the noncluster experiment to carry out the 9,000r and 12,000r series simultaneously, a control and 3,0001- dose group was performed with each of these to provide a base line dose. A 3/8 inch lucite plate covered the gelatin capsules in which the flies were contained during irradiation to provide approximate dose-depth homogeneity. For description of mutational breeding techniques in Drosophila the reader is referred to MULLER and OSTER (1963) and STURTEVANT (1956) for the prune-killer technique. RESULTS AND DISCUSSION The results of the single progeny tests seen in Table 1 and Figure 1 indicate that the sample of spermatogonia studied have lethals induced at a frequency which supports the view of proportionality to the dose over the dose range studied, 3,000 to 12,000r. The results permit the calculation of a doubling dose for spermatogonial lethals, which turns out to be about 900r for this stock under these experimental conditions. This doubling dose is in very good agreement with that cited by MULLER (1958) for both spermatogonia and oogonia. TABLE 1 Frequency of sex-linked lethals induced in spermatogonia* : I. A single drrughter tested for each treated male r 9000r 12000r Lethals - 14 (3)t 47 (8) - 56 (8) - 25 (2) No. of chromosomes tested Percent lethals 0.31 *.OS 1.25 k.i t.91 * Each PI male was mated singly and only one F, daughter was used: therefore, mutant clusters were avoided. i. The number in parentheses refers to the nuniber of near lethals, i.e. less than 10 percent survival. Dose

3 X-RAY DOSE-MUTATION FREQUENCY 359 FIGURE 1.-The I I I I DOSAGE IN KILOROENTGENS 95% CONFIDENCE LIMITS SHOWN percentage of lethals induced in spermatogonia by X rays. With the techniques employed, these studies have in all likelihood come close to the maximum feasible dose to spermatogonia because of the extremely high rate of sterility of the irradiated males. At 12,000r over 90 percent were sterile in one of the irradiations and 99 percent in another. While this undoubtedly demonstrates that the majority of spermatogonia are destroyed by the very high doses employed, it does not provide evidence for the view that sex-linked recessive lethals are differentially selected against, compared to recessive autosomal lethals. On the contrary, comparison of the sex-linked lethal frequency presented with that obtained in oogonia (MEYER and MULLER 1958) shows that they are in very good agreement, considering that errors involved in using the different stocks, X-ray sources and dosimetry are negligible. However, these experiments were not designed to test this question and are not considered conclusive. AUERBACH (1954) did attempt to answer this question and found the ratio of sex-linked to autosomal lethals lower in spermatogonia as compared with spermatozoa, but the data were not significant although she suggested that germinal selection was tak- ing place. CHANDLEY and BATEMAN (1960), on the other hand, obtained data which indicated that selection was not taking place but both experiments, because they did not avoid clustering of mutation, have large standard errors associated with the lethal frequencies. It is also of interest to note that our induced rate agrees excellently with AUERBACH S but is only one third that reported by CHAND-

4 ~~ 360 S. ABRAHAMSON AND L. D. FRIEDMAN LEY and BATEMAN. This difference in the latter case may be due to the stocks used or cell stage sampled. MOSSIGE (1956) for Canton-S reports a frequency of 1.39 i percent for 2500r X rays when cells 14 to 15 days after irradiation are tested, which is also in excellent agreement with our data. OFTEDAL (1962) has independently carried out a technically similar sex-linked lethal experiment on spermatogonia using only one off spring for each irradiated parent, as we have described. He irradiated larvae, however, with 55, 110, 160 and 300r. Almost complete killing of larvae occurs at doses in the range of 1,000r. As opposed to our results, OFTEDAL found that the frequency at the highest dose is considerably lower than expected on a simple linear relationship. In order to account for the disparity, OFTEDAL very reasonably suggests that different kinds of spermatogonia exist as has been reported for mammals; those which are both highly mutable and extremely sensitive to killing at low X-ray doses (presumably sampled by him) and the less mutable but more X-ray resistant gonial cells (presumably sampled in our experiments). Recently OFTEDAL (personal communication in manuscript) has found that very low intensity irradiations are less damaging to sensitive cells. Under these circumstances, lethals are apparently induced at a frequency proportional to dose, and these cells have a considerably higher mutation rate than those tested in our experiments. This appears to satisfy the hypothesis presented by OFTEDAL of two spermatogonial components with different mutabilities and viabilities with respect to X rays. In experiments to determine cluster size (Table 2) it can be seen that a moderate dose (for adult Drosophila) of 3,000r results in over 50 percent of the lethals occurring in clusters. These data also suggest that a considerable number of the gonial stem cells are destroyed, with subsequent repopulation by the survivors. The frequency of lethal mutation increases linearly with dose in this experiment. In both experiments (Tables 1 and 2) the mutation frequencies at 3,000r are not significantly different from each other. Since mating performance records were not kept on the males during the time required to exhaust the post-gonia1 stages, it is possible that cells other than gonia were tested. We think this possibility remote for the following reasons: (1) At the very high doses 6,000, 9,000 and 12,000r it is extremely unlikely because of their great sensitivity that any post-gonia1 cells (particularly spermatids and TABLE 2 Frequency of lethals in spermatogonia: II. Cluster size when ten progeny from each male are tested Number of lethals arising in clusters of size: Dose Number Number Perrent lethals and in roentgen P, lethals/total standard error' / t (fl probable)/ll i # 20/ f / ir I * Standard error computed by method of MULLER (1952) for mutants of common origin.

5 X-RAY DOSE-MUTATION FREQUENCY 361 spermatocytes) could have survived and resulted in viable off spring. (2) We observed a long sterile period in our cultures which corresponded to the period of spermatid and spermatocyte utilization. (3) The presence of clusters, itself is a demonstration of spermatogonial mutation. (4) The very low mutability relative to other stages attests to the fact that the vast majority of cells sampled were gonia1 as compared to the much more mutable post-gonia1 stages. This low mutability is also reflected in the high doubling dose observed. We should like to acknowledge the fine technical assistance of MRS. R. Lux, MRS. A. ROSEN- FELD, MISS E. HIMOE and MR. B. K. DAVIS in various phases of this experiment. SUMMARY Spermatogonial X-ray induced mutation rates were determined in adult Drosophila males. The mutation frequency was found to be proportional to dose for doses of 3,000 to 12,000r. LITERATURE CITED ABRAHAMSON, S., 1961 Possible repair of X-ray induced mutation in Drosophila melanogaster. (Abstr.) Genetics 46: 845. ABRAHAMSON, S., and L. FRIEDMAN, 1962 The linear relationship between X-ray dose and lethal mutations recovered from cells treated as spermatogonia in Drosophila. Proc. 2nd Intern. Congr. of Radiation Res., Harrogate p AUERBACH, C., 1954 Sensitivity of the Drosophila testis to the mutagenic action of X-rays. Z. Ind. Abst. Vererb. 86: CHANDLEY, A. C., and A. J. BATEMAN, 1960 Mutagenic sensitivity of sperm, spermatids, spermatocytes and spermatogonia in Dromphila melanogaster. Heredity, 15: MEYER, H. U,, and H. J. MULLER, 1958 Genetic effects of high doses of X-rays in oogonia. Drosophila Inform. Serv. 32: MOSSIGE, J., 1956 The relative biological efficiency of 31 Mev betatron irradiation and 175 Kev X-rays as measured by recessive sex-linked lethals in Drosophila melanogaster. Pp Progress in Radiobiology. Edited by J. S. MITCHELL, B. E. HOLMES and C. L. SMITH. Oliver and Boyd, Edinburgh. MULLER, H. J., 1952 The standard error of the frequency of mutants some of which are of common origin. (Corrected version in MULLER, H. J., 1962 Studies in Genetics. Indiana University Press, Blmmington.) Advances in radiation mutagenesis through studies on Drosophila. Proc. 2nd Intern. Conf. on Peaceful Uses of Atomic Energy, Geneva, Biological Effects of Radiation. Vol. 22: MULLER, H. J., and I. I. OSTER, 1963 Some mutational techniques in Drosophila. pp Methodology in Basic Genetics. Edited by W. J. BURDETTE. Holden-Day, San Francisco. OFTEDAL, P., 1962 Radiosensitivity of Drosophila spermatogonia. Proc. 2nd Intern. Congr. of Radiation Res., Harrogate. p RUSSELL, W. L., 1960 Dependence of mutation rate on radiation intensity. Intern. J. Rad. Biol. (suppl.) RUSSELL, W. L., L. B. RUSSELL, and E. M. KELLY, 1958 Radiation dose rate and mutation frequency. Science 128: STURTEVANT, A. H., 1956 A highly specific complementary lethal system in Drosophila melanogaster. Genetics 41: