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Dominant lethal mutations induced by MMS and mitomycin C in the fish Oryzias latipes
Mutation Research, 125 (1984) 221-227
221
Elsevier MTR 03812
Dominant lethal mutations induced by MMS and mitomycin C in the fish
Oryzias latipes A. S h i m a d a a n d N. E g a m i Zoological Institute, Faculty of Science, University of Tokyo, Hongo, Tokyo 113 (Japan) (Received 17 May 1983) (Revision received 8 September 1983) (Accepted 12 September 1983)
Summary Males of the fish Oryzias latipes were treated with various chemicals and then mated with normal females. The fertility and hatchability of the eggs laid by the parents were examined, and the dominant lethal effects were estimated. Mitomycin C induced dominant lethals in the fish spermatids and spermatocytes after the males had been treated with concentrations of 2.5 and 25 #g/ml. Methyl methanesulfonate (MMS) induced dominant lethals in spermatozoa and spermatids after the injection of 200 and 400 mg/kg. These results are in good agreement with the results obtained with mice. However, the effects of ethyl methanesulfonate (EMS) were not clear on spermatogenic ceils at any stage. We could not recognize any significant induction of dominant lethals by urethanes, bleomycin, caffeine, and two kinds of food-color additives, at least under the present experimental conditions.
The dominant lethal test with the small freshwater fish Oryzias latipes is a useful tool for detecting mutagenic factors in an aquatic environment (Egami et al., 1983). In our previous experiments, we found that: (1) among spermatogenic cells at different stages, mature sperm are most sensitive, while young germ cells are more resistant to 33-rays; (2) when mature sperm are irradiated, no dose-rate effect is observed, suggesting that young spermatogenic cells have the ability to repair themselves from sublethal damage to produce a dominant lethality; and (3) a linear relationship between the radiation dose and the hatchability decrease is recognizable within the dose range of 0-2 kR when the post-meiotic cells are irradiated. To examine the dominant lethal mutation rate after the treatment of the fish with mutagenic chemicals, and to compare the results with those obtained with 33-rays, we carried out the experiments reported herein. Male fish were treated with mitomycin C, MMS and other substances, and the 0027-5107/84/$03.00 © 1984 Elsevier Science Publishers B.V.
fertility and hatchability of eggs fertilized with the sperm produced by the treated males were examined. The results are presented here.
Materials and methods Sexually mature fish of the inbred strain (HB12) of Oryzias latipes (3-4 cm in body length) were used (Hyodo-Taguchi, 1980; Egami et al., 1983). Males were exposed to sublethal concentrations of the test compounds for given periods at 25 + 2 ° C or injected intraperitoneally with a single dose of a chemical adjusted according to the weight of the fish (which ranged from 250 to 400 mg). For the intraperitoneal injection, chemicals were dissolved in Yamamoto's Ringer solution immediately before the experiment and administered with a micro syringe (Termo). Table 1 shows the chemicals tested and the methods used for the treatment of the fish. Immediately after the treatment, each male was
222 TABLE 1 CHEMICALS TESTED FOR INDUCTION OF DOMINANT LETHALITY Compound Mitomycin C Bleomycin Urethane
Concn. in water (# g/ml) 25 2.5
1000 100
Phenyhrethane
100 10
Caffeine Newco~ine
Dose (mg/kg)
24 24
20
Source (name of company) Kyowa Hakkou
30 1000 100
Methylurethane
Exposed period (h)
24 72 24 72 24 72 24 72 24 72 24 72 24
Nihon Kayaku Tokyo Kasei
Tokyo Kasei
Tokyo Kasei
200 100 320 160 400 200 1600
Tartrazine MMS (methyl methanesulfonate) EMS (ethyl methanesulfonate)
Tokyo Kasei Tokyo Kasei Tokyo Kasei Nakarai Chemicals Nakarai Chemicals
800 400 800 200
kept, together with an intact female, in a vessel containing about 1 1 of water. Each pair was kept in a culture room (25-29°C; 14 h of light per day; Tetra-Min food, Tetra Werke, West Germany) during the course of the examination. Under such conditions, most of the pairs mated, and clusters of eggs (each containing 10-40 eggs) were laid almost every day beginning the next morning. The eggs were counted every day in order to estimate the fertility of the males. Each duster of fertilized eggs was cultured at 25-29°C in a petri dish 9 cm in diameter, and the number of dead embryos and newly hatched fry were counted in order to calculate the hatchability (Egami et al., 1983). The fertility and hatchability of the non-treated control fish were 96 + 1.9 and 98 + 1.1% respectively (Table 4). The times required for all the gametogenetic stages to develop into mature sperm had previ-
2
2
ously been determined by Egami and others (Egami and Hyodo-Taguchi, 1967; Egami et al., 1976). If the development of the spermatogenetic cells is not affected by the treatment, the exposed stages of spermatogenetic cells can be predicted, from the interval between treatment and fertilization, to be as follows: spermatozoa, 1-4 days; spermatids, 4-9 days; and spermatocytes, 9-13 days. Therefore, the brood pattern of the hatchability was examined. Results
Mitomycin C The results of the mitomycin C experiment are summarized in Table 2. A considerable reduction in the hatchability of the eggs was observed if the eggs were fertilized 4-13 days after treatment with rnitomycin C, 25 #g/ml. The number of pairs was small, and the fluctuation in the hatchability was
223 TABLE 2 EFFECTS OF MITOMYCIN C ON FERTILITY AND HATCHABILITY Interval between treatment and fertilization (days)
Number of eggs obtained
Number of fertilized eggs
Fertility (% 4- S.E.)
3
1- 4 4- 9 9-13 13-47
225 381 233 1330
221 369 220 989
98+ 0.6 97+ 0.6 94+ 2.9 74+ 11.8
156 131 67 847
71+ 36+ 31-186+
3
1- 4 4- 9 9-13 13-47
172 314 249 841
142 291 233 551
83 + 8.1 93+ 4.1 944- 0.6 66 4- 21.2
86 146 144 407
61 + 16.0 55+27.0 61 4-27.0 87 4-13.3
Concn. in water (#g/ml)
Number of pairs
25
2.5
large in the groups treated with 2.5 #g of mitomycin C per ml. The brood pattern of the hatchability is shown in Fig. 1. The hatchability began to decrease just after treatment, falling to its lowest level in about 5-10 days, and then rapidly increasing to 80% of the control 20 days after the treatment. Thereafter, taking over 30 days, it continued to recover toward to non-treated level. The reduction in fertility, which was taken as an indicator of the killing effect on male germ cells, was only observed 13-47 days after the treatment (Table 2). Fig. 2 shows the change in the fertility after treatment with 25 #g of mitomycin C
Number of hatched
Hatehability (% 4-S.E.)
fry 6.9 5.4 8.8 5.0
p e r nil. T h e fertility b eg an to decrease at a b o u t the 10th day after the treatment, b u t f r o m a b o u t the 20th day it gradually r eco v er ed to the c o n t r o l level. F r o m these results it seems p r o b a b l e that m i t o m y c i n C i n d u c e d a high f r e q u e n c y o f d o m i n a n t lethal m u t a t i o n s in fish spermatids and spermatocytes, a n d that s p e r m a t o g o n i a were the germ-cell stage m o s t sensitive to the cell-killing effect of this chemical.
MMS After the injection of male fish with 400 and
%100
- •
@ 00-
0
00
0 0
°\T Oo./ @\@
0
@
/
•
lb
2b DAYS
AFTER
--
- -
•
25.0
a'o
~g/rnt
3'5
4b
TREATMENTS
Fig. 1. The brood pattern of the hatcbability of embryos fertilized with sperm of males treated with mitomycin C. Males were exposed to water containing mitomycin C at concentrations of 25 and 2.5 #g/ml for 24 h at 25 5: 2°C.
224
%1oo J
p. =:50 u.I tl.
25.0pg/mt l
o
5
I
1
10
15
1
20
25
DAYS AFTER
30
I
35
40
45
TREATMENTS
Fig. 2. Change in the fertility of males treated with mitomycin C, 25 # g / m l .
200 mg/kg doses of MMS, the frequency of the death of embryos fertilized with the male was very high for the first 9 days (Table 3). A linear dose-effect relationship was recognizable 2-9 days after the injection with 200 and 400 mg/kg doses of MMS (Fig. 3). The change in the hatchability of these embryos is shown in Fig. 4. The hatchability decreased markedly 2 days after the treatment (400 mg/kg); this low level of hatchability continued for several days, and then it began to increase and recovered to the control level within 20 days after the treatment. These results indicate that MMS induced dominant lethals, especially in the spermatozoa and spermatids of the fish.
A reduction in fertility was also observed after treatment with both doses of MMS (Table 3). Therefore, spermatogonia may be killed by MMS. EMS
In the preliminary experiments, male fish were exposed to concentrations of 800 and 200 /~g of EMS per ml for 2 h; no significant induction of dominant lethal mutation was observed. Then males were injected with 400, 800 and 1600 mg/kg doses of EMS. Even at 800 mg/kg, most of the fish were killed. The number of males that survived was small; however, no significant decrease in hatchability was recognizable with any dose (Table 4, Fig. 4).
TABLE 3 EFFECTS O F MMS ON FERTILITY A N D H A T C H A B I L I T Y Dose (mg/kg)
Number of pairs
Interval between treatment and fertilization (days)
Number of eggs obtained
Number of fertilized eggs
Fertility (% _+S.E.)
Number of hatched fry
Hatchability (% + S.E.)
400
6
1- 4 4- 9 9-13 13-19
300 476 238 256
263 351 134 150
88 + 73+ 56 + 595-
6.3 7.6 12.8 6.0
91 97 73 115
35 + 8,7 28 ± 7,4 54 +_8.9 77+3.5
200
6
1- 4 4- 9 9-13 13-19
426 652 399 339
398 632 346 284
93 + 97 -l87 + 84+
2.9 2.2 2.9 7.1
258 368 273 255
65 + 5.0 58 + 6.2 79 + 5.4 90+4.0
225 10C
100
A
Z < "1- 5[
L l
"~
~
', ', ,,
,o."
~
6
260
)~,~/ ~
5 Doys after -- sperm-- -tids --
460
Dose (mg/kg)
'
x,"
~
< "r"
r .....7
..o"
p,".,
10 treotment -- cytes--
•
control
• ~
400mg/kgMMS 800mg/kgEMS
20
15 --
gonio --
Fig. 3. Dose-effect relationship of the hatchability of embryos fertilized 2 - 9 days after the injection of males with MMS. Vertical lines indicate standard errors of the mean.
Fig. 4. The brood pattern of the hatchability of embryo~ fertilized with sperm of males treated with MMS (200, 4(,~, m g / k g ) and EMS (800 m g / k g ) .
Bleomycin and other chemicals No dominant lethality was observed in any spermatogenic cells after injection with the maximal sublethal dose of bleomycin (30 mg/kg). Exposure of the male to a maximal non-lethal concentration of caffeine (20/,g/ml) for 24 h did not decrease the hatchability. Furthermore, urethane and methylurethane (1000, 100/~g/ml; for 24, 72 h), as well as phenylurethane (100, 10/~g/ml; for 24, 72 h), induced neither dominant lethals nor any killing of germ cells. Two kinds of edible dye, newcoccine (red No. 102) and tartrazine (yellow No. 4), were injected into males but they did not at all affect the hatchability of the embryos fertilized with sperm of the males.
Discussion
It has already been established that, when males of the fish Oryzias latipes are irradiated with y-rays, the hatchability of eggs fertilized with sperm of the irradiated males is reduced, and that the reduction in the rate of the hatchability is dependent on the irradiated stage of the spermatogenetic cells (Sakaizumi and Egami, 1980; Egami et al., 1983). In other words, ionizing radiation induces dominant lethal mutations in spermatogenetic cells. If the interval between irradiation and fertilization is longer than 50 days, however, the effects become smaller. On the basis of these results, we have suggested that the young spermatogenic cells are resistant to y-rays.
TABLE 4 E F F E C T O F EMS ON F E R T I L I T Y A N D H A T C H A B I L I T Y Dose (mg/kg)
Number of pairs
Interval between treatment and fertilization (days)
Number of eggs obtained
Number of fertilized eggs
Fertility (% 5: S.E.)
Number of hatched fry
Hatchability (% 5: S.E.)
1600
2
1- 4 4- 9 9-13 13-19
78 147 137 110
32 130 126 102
41 + 88+ 92+ 93+
34.0 7.5 4.0 9.5
27 118 117 96
84 + 91+ 93+ 94+
31.0 7.0 2.0 6.5
800
4
1- 4 4- 9 9-13 13-19
188 315 190 174
168 299 183 164
89-1- 2.6 95 + 2.0 965:1.6 9 4 + 1.3
159 290 175 157
95+ 97 + 96+ 96+
6.2 1.8 3.4 2.1
Control
14
1-19
914
877
965: 1.9
858
985: 1.1
226
The present experiments were designed to compare the effects of )'-rays and those of chemicals on the induction of dominant lethal mutations in spermatogenetic cells at different stages. Chemicals that have different modes of action were selected for the present experiments. Mitomycin C is said to inhibit DNA synthesis by the cross-linking of complementary strands of DNA (Iyer and Szybalski, 1964). Both MMS and EMS are alkylating agents that react with the DNA bases; it has been reported that MMS is about 4 times as effective as EMS in inducing dominant lethality in mouse spermatozoa and late spermatids (Ehling et al., 1968). The carcinogenic action of urethane is well known in mammals, and the teratogenic action of this chemical has been found in Oryzias embryos (unpublished data). It is difficult to predict the exact stages of the spermatogenetic cells exposed to chemicals because chemicals disturb the normal development of germ cells. Particularly in fish, mature sperm at various spermatogenic stages at the time of the treatment are mixed to some extent during storage for 1-5 days in the center of the testis. Taking this fact into account, it appears from the present experiments on hatchability that the sensitive spermatogenic cells in the induction of dominant lethals by mitomycin C and MMS are different; the former are spermatids and spermatocytes, while the latter are spermatozoa and spermatids. In the mouse, the yields of radiation-induced dominant lethal mutations in early spermatids are two or three times the mutation yields in spermatozoa, late spermatids and spermatocytes (Bateman, 1958; Ehling, 1971). In the fish Oryzias latipes, however, the most sensitive stage to the induction of dominant lethals by radiation is that of the spermatozoa (Egami and Hyodo-Taguchi, 1973; Sakaizumi and Egami, 1980; Egami et al., 1983). A similar difference in stage sensitivity between mitomycin C and MMS has been found in the mouse (Ehling et al., 1968; Ehling, 1971). One of the most noticeable things is that EMS, which induces more than 90% of the dominant lethal mutations in the mouse late spermatids after the injection of EMS, 400 mg/kg, had no effect on the induction of dominant lethality of the fish Oryzias latipes even if 1600 mg/kg was injected into males. Sega (1980) and Sega and Owens (1978)
investigated the molecular dosimetry of EMS in the germ cells of male mice and presented the following model to explain how dominant lethals occur during the treatment of EMS. After protamine synthesis (in maturing spermatid stages), but before cysteine disulfide bonds are formed, EMS ethylates the sulfhydryl groups of protamine (not of the DNA) and thus effectively blocks normal formation of disulfide bonds. This, in turn, would prevent normal chromatin condensation in the sperm nucleus, leading to stresses in the chromatin structure that would eventually result in a lethal chromosome break. Cysteine, the target for alkylation by EMS, is said to be present in the sperm protamine of all eytherian mammals so far studied; it represents about 10% of the amino acids in the protein (Bellv~ et al., 1975). In contrast, in a great many species of fish, protamine does not contain cysteine (Hnilica, 1972). In Oryzias latipes, the amino-acid composition of basic proteins in the sperm nuclei has not yet been studied, but if this species also has a protamine including no cysteine, and if the model of the mechanism for the action by EMS presented by Sega is true, our finding that EMS did not significantly induce dominant lethals in Oryzias latipes seems to be reasonable. Killing effects of chemicals on spermatogenetic cells were observed in terms of the proportion of fertilized eggs. This was confirmed by the histology of the testis (Egami et al., 1983). The pattern of the killing effects of ),-rays was similar to those of several chemicals. The fertility of males irradiated with 2 kR remains unchanged for about the first 10 days, but it decreases for the next 4 weeks or so (Konno and Egami, 1966; Sakaizumi and Egami, 1980; Egami et al., 1983). A similar pattern was found in the effect of mitomycin C (Fig. 2). From the time course of the changes in fertility, the cause of the reduction by mitomycin C as well as by y-rays may be the reduction in the number of premeiotic spermatogenic cells (spermatogonia Ib, spermatogonia II, and spermatocytes in the meiotic prophase). The stem cells of germ cells, on the other hand, might be resistant to mitomycin C, for the fertility finally recovered after a temporary sterilization. The reduction in fertility 9-19 days after treatment with MMS also appears to be due to the killing effect of MMS on premeiotic germ
227 cells, like other m u t a g e n i c factors. The action of chemicals o n the i n d u c t i o n of d o m i n a n t lethal m u t a t i o n a n d o n cell lethality is p r o b a b l y different from these results. Acknowledgements This work was supported in part b y a G r a n t - i n A i d for E n v i r o n m e n t a l Science from the M i n i s t r y of Education, Science, a n d C u l t u r e to N.E. We t h a n k Dr. Y. H y o d o - T a g u c h i of the N a t i o n a l Institute of Radiological Sciences for her k i n d supply of the HB1-2 i n b r e d strain of Oryzias latipes. We also t h a n k Miss K. U m e n o , Miss Y. K i n o s h i t a a n d Miss Y. T o m a b e c h i for their technical assistance d u r i n g the course of the experiments.
References Bateman, A.J. (1958) Mutagenic sensitivity of maturing germ cells in the male mouse, Heredity, 12, 213-232. Bellvr, A.R., E. Anderson and L. Hanley-Bowdoin (1975) Synthesis and amino acid composition of basic proteins in mammalian sperm nuclei, Develop. Biol., 47, 349-365. Egami, N., and Y. Hyodo-Taguchi (1967) An autoradiographic examination of rate of spermatogenesisat different temperatures in the fish, Oryzias latipes, Exp. Cell Res., 47, 665-667. Egami, N., and Y. Hyodo-Taguchi (1973) Dominant lethal mutation rate in the fish, Oryzias latipes, irradiated at various stages of gametogenesis, in: J.H. Schr~kler (Ed.), Genetics and Mutagenesis in Fish, Springer, Berlin, pp. 75-81.
Egami, N., Y. Hyodo-Taguchi and K. Konno (1976) Autoradiographical studies of spermatogenesisin the fish, Oryzias latipes, with special reference to radiation effects on the fertility of fish, Gunma Symp. Endocrinol., 4, 147-159. Egami, N., A. Shimada and A. Hama-Furukawa (1983) Dominant lethal mutation rate after y-irradiation of the fish, Oryzias latipes, Mutation Res., 107, 265-277. Ehling, U.H. (1971) Comparison of radiation- and chemicallyinduced dominant lethal mutations in male mice, Mutation Res., 11, 35-44. Ehling, U.H., R.B. Cumming and H.V. Malting (1968) Induction of dominant lethal mutations by alkylating agents in mate mice, Mutation Res., 5, 417-428. Hnilica, L.S. (1972) The structure and biological function of histone, CRC Press, Ohio City, Chapter 2. Hyodo-Taguchi, Y. (1980) Establishment of inbred strains of the teleost, Oryzias latipes, Zooi. Mag. (Tokyo), 89, 283-301. Iyer, V.N., and W. Szybalski (1964) Mitomycins and porfiromycin; Chemical mechanism of activation and cross-linkingof DNA, Science, 145, 55-58. Konno, K., and N. Egami (1966) Notes on effects of X-irradiation on the fertifity of the male of Oryzias latipes (Teleostei, Cyprinodontidae), Annot. Zool. Jpn., 39, 63-70. Sakaizumi, M., and N. Egami (1980) Effects of methyl mercuric chloride and gamma irradiation on the fertility of males in the fish, Oryzias latipes, J. Fac. Sci., Univ. Tokyo, IV, 14, 385-390. Sega, G.A. (1980) Relationship between unscheduled DNA synthesis and mutation induction in male mice, in: W.M. Generoso (Ed.), DNA Repair and Mutagenesis in Eukaryotes, Plenum, New York, pp. 373-383. Sega, G.A., and J.G. Owens (1978) Ethylation of DNA and protamine by ethyl methanesulfonate in the germ cells of male mice and the relevancy of these molecular targets to the induction of dominant lethals, Mutation Res., 52, 87-106.
221
Elsevier MTR 03812
Dominant lethal mutations induced by MMS and mitomycin C in the fish
Oryzias latipes A. S h i m a d a a n d N. E g a m i Zoological Institute, Faculty of Science, University of Tokyo, Hongo, Tokyo 113 (Japan) (Received 17 May 1983) (Revision received 8 September 1983) (Accepted 12 September 1983)
Summary Males of the fish Oryzias latipes were treated with various chemicals and then mated with normal females. The fertility and hatchability of the eggs laid by the parents were examined, and the dominant lethal effects were estimated. Mitomycin C induced dominant lethals in the fish spermatids and spermatocytes after the males had been treated with concentrations of 2.5 and 25 #g/ml. Methyl methanesulfonate (MMS) induced dominant lethals in spermatozoa and spermatids after the injection of 200 and 400 mg/kg. These results are in good agreement with the results obtained with mice. However, the effects of ethyl methanesulfonate (EMS) were not clear on spermatogenic ceils at any stage. We could not recognize any significant induction of dominant lethals by urethanes, bleomycin, caffeine, and two kinds of food-color additives, at least under the present experimental conditions.
The dominant lethal test with the small freshwater fish Oryzias latipes is a useful tool for detecting mutagenic factors in an aquatic environment (Egami et al., 1983). In our previous experiments, we found that: (1) among spermatogenic cells at different stages, mature sperm are most sensitive, while young germ cells are more resistant to 33-rays; (2) when mature sperm are irradiated, no dose-rate effect is observed, suggesting that young spermatogenic cells have the ability to repair themselves from sublethal damage to produce a dominant lethality; and (3) a linear relationship between the radiation dose and the hatchability decrease is recognizable within the dose range of 0-2 kR when the post-meiotic cells are irradiated. To examine the dominant lethal mutation rate after the treatment of the fish with mutagenic chemicals, and to compare the results with those obtained with 33-rays, we carried out the experiments reported herein. Male fish were treated with mitomycin C, MMS and other substances, and the 0027-5107/84/$03.00 © 1984 Elsevier Science Publishers B.V.
fertility and hatchability of eggs fertilized with the sperm produced by the treated males were examined. The results are presented here.
Materials and methods Sexually mature fish of the inbred strain (HB12) of Oryzias latipes (3-4 cm in body length) were used (Hyodo-Taguchi, 1980; Egami et al., 1983). Males were exposed to sublethal concentrations of the test compounds for given periods at 25 + 2 ° C or injected intraperitoneally with a single dose of a chemical adjusted according to the weight of the fish (which ranged from 250 to 400 mg). For the intraperitoneal injection, chemicals were dissolved in Yamamoto's Ringer solution immediately before the experiment and administered with a micro syringe (Termo). Table 1 shows the chemicals tested and the methods used for the treatment of the fish. Immediately after the treatment, each male was
222 TABLE 1 CHEMICALS TESTED FOR INDUCTION OF DOMINANT LETHALITY Compound Mitomycin C Bleomycin Urethane
Concn. in water (# g/ml) 25 2.5
1000 100
Phenyhrethane
100 10
Caffeine Newco~ine
Dose (mg/kg)
24 24
20
Source (name of company) Kyowa Hakkou
30 1000 100
Methylurethane
Exposed period (h)
24 72 24 72 24 72 24 72 24 72 24 72 24
Nihon Kayaku Tokyo Kasei
Tokyo Kasei
Tokyo Kasei
200 100 320 160 400 200 1600
Tartrazine MMS (methyl methanesulfonate) EMS (ethyl methanesulfonate)
Tokyo Kasei Tokyo Kasei Tokyo Kasei Nakarai Chemicals Nakarai Chemicals
800 400 800 200
kept, together with an intact female, in a vessel containing about 1 1 of water. Each pair was kept in a culture room (25-29°C; 14 h of light per day; Tetra-Min food, Tetra Werke, West Germany) during the course of the examination. Under such conditions, most of the pairs mated, and clusters of eggs (each containing 10-40 eggs) were laid almost every day beginning the next morning. The eggs were counted every day in order to estimate the fertility of the males. Each duster of fertilized eggs was cultured at 25-29°C in a petri dish 9 cm in diameter, and the number of dead embryos and newly hatched fry were counted in order to calculate the hatchability (Egami et al., 1983). The fertility and hatchability of the non-treated control fish were 96 + 1.9 and 98 + 1.1% respectively (Table 4). The times required for all the gametogenetic stages to develop into mature sperm had previ-
2
2
ously been determined by Egami and others (Egami and Hyodo-Taguchi, 1967; Egami et al., 1976). If the development of the spermatogenetic cells is not affected by the treatment, the exposed stages of spermatogenetic cells can be predicted, from the interval between treatment and fertilization, to be as follows: spermatozoa, 1-4 days; spermatids, 4-9 days; and spermatocytes, 9-13 days. Therefore, the brood pattern of the hatchability was examined. Results
Mitomycin C The results of the mitomycin C experiment are summarized in Table 2. A considerable reduction in the hatchability of the eggs was observed if the eggs were fertilized 4-13 days after treatment with rnitomycin C, 25 #g/ml. The number of pairs was small, and the fluctuation in the hatchability was
223 TABLE 2 EFFECTS OF MITOMYCIN C ON FERTILITY AND HATCHABILITY Interval between treatment and fertilization (days)
Number of eggs obtained
Number of fertilized eggs
Fertility (% 4- S.E.)
3
1- 4 4- 9 9-13 13-47
225 381 233 1330
221 369 220 989
98+ 0.6 97+ 0.6 94+ 2.9 74+ 11.8
156 131 67 847
71+ 36+ 31-186+
3
1- 4 4- 9 9-13 13-47
172 314 249 841
142 291 233 551
83 + 8.1 93+ 4.1 944- 0.6 66 4- 21.2
86 146 144 407
61 + 16.0 55+27.0 61 4-27.0 87 4-13.3
Concn. in water (#g/ml)
Number of pairs
25
2.5
large in the groups treated with 2.5 #g of mitomycin C per ml. The brood pattern of the hatchability is shown in Fig. 1. The hatchability began to decrease just after treatment, falling to its lowest level in about 5-10 days, and then rapidly increasing to 80% of the control 20 days after the treatment. Thereafter, taking over 30 days, it continued to recover toward to non-treated level. The reduction in fertility, which was taken as an indicator of the killing effect on male germ cells, was only observed 13-47 days after the treatment (Table 2). Fig. 2 shows the change in the fertility after treatment with 25 #g of mitomycin C
Number of hatched
Hatehability (% 4-S.E.)
fry 6.9 5.4 8.8 5.0
p e r nil. T h e fertility b eg an to decrease at a b o u t the 10th day after the treatment, b u t f r o m a b o u t the 20th day it gradually r eco v er ed to the c o n t r o l level. F r o m these results it seems p r o b a b l e that m i t o m y c i n C i n d u c e d a high f r e q u e n c y o f d o m i n a n t lethal m u t a t i o n s in fish spermatids and spermatocytes, a n d that s p e r m a t o g o n i a were the germ-cell stage m o s t sensitive to the cell-killing effect of this chemical.
MMS After the injection of male fish with 400 and
%100
- •
@ 00-
0
00
0 0
°\T Oo./ @\@
0
@
/
•
lb
2b DAYS
AFTER
--
- -
•
25.0
a'o
~g/rnt
3'5
4b
TREATMENTS
Fig. 1. The brood pattern of the hatcbability of embryos fertilized with sperm of males treated with mitomycin C. Males were exposed to water containing mitomycin C at concentrations of 25 and 2.5 #g/ml for 24 h at 25 5: 2°C.
224
%1oo J
p. =:50 u.I tl.
25.0pg/mt l
o
5
I
1
10
15
1
20
25
DAYS AFTER
30
I
35
40
45
TREATMENTS
Fig. 2. Change in the fertility of males treated with mitomycin C, 25 # g / m l .
200 mg/kg doses of MMS, the frequency of the death of embryos fertilized with the male was very high for the first 9 days (Table 3). A linear dose-effect relationship was recognizable 2-9 days after the injection with 200 and 400 mg/kg doses of MMS (Fig. 3). The change in the hatchability of these embryos is shown in Fig. 4. The hatchability decreased markedly 2 days after the treatment (400 mg/kg); this low level of hatchability continued for several days, and then it began to increase and recovered to the control level within 20 days after the treatment. These results indicate that MMS induced dominant lethals, especially in the spermatozoa and spermatids of the fish.
A reduction in fertility was also observed after treatment with both doses of MMS (Table 3). Therefore, spermatogonia may be killed by MMS. EMS
In the preliminary experiments, male fish were exposed to concentrations of 800 and 200 /~g of EMS per ml for 2 h; no significant induction of dominant lethal mutation was observed. Then males were injected with 400, 800 and 1600 mg/kg doses of EMS. Even at 800 mg/kg, most of the fish were killed. The number of males that survived was small; however, no significant decrease in hatchability was recognizable with any dose (Table 4, Fig. 4).
TABLE 3 EFFECTS O F MMS ON FERTILITY A N D H A T C H A B I L I T Y Dose (mg/kg)
Number of pairs
Interval between treatment and fertilization (days)
Number of eggs obtained
Number of fertilized eggs
Fertility (% _+S.E.)
Number of hatched fry
Hatchability (% + S.E.)
400
6
1- 4 4- 9 9-13 13-19
300 476 238 256
263 351 134 150
88 + 73+ 56 + 595-
6.3 7.6 12.8 6.0
91 97 73 115
35 + 8,7 28 ± 7,4 54 +_8.9 77+3.5
200
6
1- 4 4- 9 9-13 13-19
426 652 399 339
398 632 346 284
93 + 97 -l87 + 84+
2.9 2.2 2.9 7.1
258 368 273 255
65 + 5.0 58 + 6.2 79 + 5.4 90+4.0
225 10C
100
A
Z < "1- 5[
L l
"~
~
', ', ,,
,o."
~
6
260
)~,~/ ~
5 Doys after -- sperm-- -tids --
460
Dose (mg/kg)
'
x,"
~
< "r"
r .....7
..o"
p,".,
10 treotment -- cytes--
•
control
• ~
400mg/kgMMS 800mg/kgEMS
20
15 --
gonio --
Fig. 3. Dose-effect relationship of the hatchability of embryos fertilized 2 - 9 days after the injection of males with MMS. Vertical lines indicate standard errors of the mean.
Fig. 4. The brood pattern of the hatchability of embryo~ fertilized with sperm of males treated with MMS (200, 4(,~, m g / k g ) and EMS (800 m g / k g ) .
Bleomycin and other chemicals No dominant lethality was observed in any spermatogenic cells after injection with the maximal sublethal dose of bleomycin (30 mg/kg). Exposure of the male to a maximal non-lethal concentration of caffeine (20/,g/ml) for 24 h did not decrease the hatchability. Furthermore, urethane and methylurethane (1000, 100/~g/ml; for 24, 72 h), as well as phenylurethane (100, 10/~g/ml; for 24, 72 h), induced neither dominant lethals nor any killing of germ cells. Two kinds of edible dye, newcoccine (red No. 102) and tartrazine (yellow No. 4), were injected into males but they did not at all affect the hatchability of the embryos fertilized with sperm of the males.
Discussion
It has already been established that, when males of the fish Oryzias latipes are irradiated with y-rays, the hatchability of eggs fertilized with sperm of the irradiated males is reduced, and that the reduction in the rate of the hatchability is dependent on the irradiated stage of the spermatogenetic cells (Sakaizumi and Egami, 1980; Egami et al., 1983). In other words, ionizing radiation induces dominant lethal mutations in spermatogenetic cells. If the interval between irradiation and fertilization is longer than 50 days, however, the effects become smaller. On the basis of these results, we have suggested that the young spermatogenic cells are resistant to y-rays.
TABLE 4 E F F E C T O F EMS ON F E R T I L I T Y A N D H A T C H A B I L I T Y Dose (mg/kg)
Number of pairs
Interval between treatment and fertilization (days)
Number of eggs obtained
Number of fertilized eggs
Fertility (% 5: S.E.)
Number of hatched fry
Hatchability (% 5: S.E.)
1600
2
1- 4 4- 9 9-13 13-19
78 147 137 110
32 130 126 102
41 + 88+ 92+ 93+
34.0 7.5 4.0 9.5
27 118 117 96
84 + 91+ 93+ 94+
31.0 7.0 2.0 6.5
800
4
1- 4 4- 9 9-13 13-19
188 315 190 174
168 299 183 164
89-1- 2.6 95 + 2.0 965:1.6 9 4 + 1.3
159 290 175 157
95+ 97 + 96+ 96+
6.2 1.8 3.4 2.1
Control
14
1-19
914
877
965: 1.9
858
985: 1.1
226
The present experiments were designed to compare the effects of )'-rays and those of chemicals on the induction of dominant lethal mutations in spermatogenetic cells at different stages. Chemicals that have different modes of action were selected for the present experiments. Mitomycin C is said to inhibit DNA synthesis by the cross-linking of complementary strands of DNA (Iyer and Szybalski, 1964). Both MMS and EMS are alkylating agents that react with the DNA bases; it has been reported that MMS is about 4 times as effective as EMS in inducing dominant lethality in mouse spermatozoa and late spermatids (Ehling et al., 1968). The carcinogenic action of urethane is well known in mammals, and the teratogenic action of this chemical has been found in Oryzias embryos (unpublished data). It is difficult to predict the exact stages of the spermatogenetic cells exposed to chemicals because chemicals disturb the normal development of germ cells. Particularly in fish, mature sperm at various spermatogenic stages at the time of the treatment are mixed to some extent during storage for 1-5 days in the center of the testis. Taking this fact into account, it appears from the present experiments on hatchability that the sensitive spermatogenic cells in the induction of dominant lethals by mitomycin C and MMS are different; the former are spermatids and spermatocytes, while the latter are spermatozoa and spermatids. In the mouse, the yields of radiation-induced dominant lethal mutations in early spermatids are two or three times the mutation yields in spermatozoa, late spermatids and spermatocytes (Bateman, 1958; Ehling, 1971). In the fish Oryzias latipes, however, the most sensitive stage to the induction of dominant lethals by radiation is that of the spermatozoa (Egami and Hyodo-Taguchi, 1973; Sakaizumi and Egami, 1980; Egami et al., 1983). A similar difference in stage sensitivity between mitomycin C and MMS has been found in the mouse (Ehling et al., 1968; Ehling, 1971). One of the most noticeable things is that EMS, which induces more than 90% of the dominant lethal mutations in the mouse late spermatids after the injection of EMS, 400 mg/kg, had no effect on the induction of dominant lethality of the fish Oryzias latipes even if 1600 mg/kg was injected into males. Sega (1980) and Sega and Owens (1978)
investigated the molecular dosimetry of EMS in the germ cells of male mice and presented the following model to explain how dominant lethals occur during the treatment of EMS. After protamine synthesis (in maturing spermatid stages), but before cysteine disulfide bonds are formed, EMS ethylates the sulfhydryl groups of protamine (not of the DNA) and thus effectively blocks normal formation of disulfide bonds. This, in turn, would prevent normal chromatin condensation in the sperm nucleus, leading to stresses in the chromatin structure that would eventually result in a lethal chromosome break. Cysteine, the target for alkylation by EMS, is said to be present in the sperm protamine of all eytherian mammals so far studied; it represents about 10% of the amino acids in the protein (Bellv~ et al., 1975). In contrast, in a great many species of fish, protamine does not contain cysteine (Hnilica, 1972). In Oryzias latipes, the amino-acid composition of basic proteins in the sperm nuclei has not yet been studied, but if this species also has a protamine including no cysteine, and if the model of the mechanism for the action by EMS presented by Sega is true, our finding that EMS did not significantly induce dominant lethals in Oryzias latipes seems to be reasonable. Killing effects of chemicals on spermatogenetic cells were observed in terms of the proportion of fertilized eggs. This was confirmed by the histology of the testis (Egami et al., 1983). The pattern of the killing effects of ),-rays was similar to those of several chemicals. The fertility of males irradiated with 2 kR remains unchanged for about the first 10 days, but it decreases for the next 4 weeks or so (Konno and Egami, 1966; Sakaizumi and Egami, 1980; Egami et al., 1983). A similar pattern was found in the effect of mitomycin C (Fig. 2). From the time course of the changes in fertility, the cause of the reduction by mitomycin C as well as by y-rays may be the reduction in the number of premeiotic spermatogenic cells (spermatogonia Ib, spermatogonia II, and spermatocytes in the meiotic prophase). The stem cells of germ cells, on the other hand, might be resistant to mitomycin C, for the fertility finally recovered after a temporary sterilization. The reduction in fertility 9-19 days after treatment with MMS also appears to be due to the killing effect of MMS on premeiotic germ
227 cells, like other m u t a g e n i c factors. The action of chemicals o n the i n d u c t i o n of d o m i n a n t lethal m u t a t i o n a n d o n cell lethality is p r o b a b l y different from these results. Acknowledgements This work was supported in part b y a G r a n t - i n A i d for E n v i r o n m e n t a l Science from the M i n i s t r y of Education, Science, a n d C u l t u r e to N.E. We t h a n k Dr. Y. H y o d o - T a g u c h i of the N a t i o n a l Institute of Radiological Sciences for her k i n d supply of the HB1-2 i n b r e d strain of Oryzias latipes. We also t h a n k Miss K. U m e n o , Miss Y. K i n o s h i t a a n d Miss Y. T o m a b e c h i for their technical assistance d u r i n g the course of the experiments.
References Bateman, A.J. (1958) Mutagenic sensitivity of maturing germ cells in the male mouse, Heredity, 12, 213-232. Bellvr, A.R., E. Anderson and L. Hanley-Bowdoin (1975) Synthesis and amino acid composition of basic proteins in mammalian sperm nuclei, Develop. Biol., 47, 349-365. Egami, N., and Y. Hyodo-Taguchi (1967) An autoradiographic examination of rate of spermatogenesisat different temperatures in the fish, Oryzias latipes, Exp. Cell Res., 47, 665-667. Egami, N., and Y. Hyodo-Taguchi (1973) Dominant lethal mutation rate in the fish, Oryzias latipes, irradiated at various stages of gametogenesis, in: J.H. Schr~kler (Ed.), Genetics and Mutagenesis in Fish, Springer, Berlin, pp. 75-81.
Egami, N., Y. Hyodo-Taguchi and K. Konno (1976) Autoradiographical studies of spermatogenesisin the fish, Oryzias latipes, with special reference to radiation effects on the fertility of fish, Gunma Symp. Endocrinol., 4, 147-159. Egami, N., A. Shimada and A. Hama-Furukawa (1983) Dominant lethal mutation rate after y-irradiation of the fish, Oryzias latipes, Mutation Res., 107, 265-277. Ehling, U.H. (1971) Comparison of radiation- and chemicallyinduced dominant lethal mutations in male mice, Mutation Res., 11, 35-44. Ehling, U.H., R.B. Cumming and H.V. Malting (1968) Induction of dominant lethal mutations by alkylating agents in mate mice, Mutation Res., 5, 417-428. Hnilica, L.S. (1972) The structure and biological function of histone, CRC Press, Ohio City, Chapter 2. Hyodo-Taguchi, Y. (1980) Establishment of inbred strains of the teleost, Oryzias latipes, Zooi. Mag. (Tokyo), 89, 283-301. Iyer, V.N., and W. Szybalski (1964) Mitomycins and porfiromycin; Chemical mechanism of activation and cross-linkingof DNA, Science, 145, 55-58. Konno, K., and N. Egami (1966) Notes on effects of X-irradiation on the fertifity of the male of Oryzias latipes (Teleostei, Cyprinodontidae), Annot. Zool. Jpn., 39, 63-70. Sakaizumi, M., and N. Egami (1980) Effects of methyl mercuric chloride and gamma irradiation on the fertility of males in the fish, Oryzias latipes, J. Fac. Sci., Univ. Tokyo, IV, 14, 385-390. Sega, G.A. (1980) Relationship between unscheduled DNA synthesis and mutation induction in male mice, in: W.M. Generoso (Ed.), DNA Repair and Mutagenesis in Eukaryotes, Plenum, New York, pp. 373-383. Sega, G.A., and J.G. Owens (1978) Ethylation of DNA and protamine by ethyl methanesulfonate in the germ cells of male mice and the relevancy of these molecular targets to the induction of dominant lethals, Mutation Res., 52, 87-106.