Proc. Nati. Acad. Sci. USAVol. 88, pp. 2545-2549, March 1991Genetics

Development of a possible nonmammalian test system forradiation-induced germ-cell mutagenesis using a fish,the Japanese medaka (Oryzias latipes)

(specific-locus test/total mutation/viable mutation/dolant lethal/teleost)

AKIHIRO SHIMA* AND ATSUKO SHIMADALaboratory of Radiation Biology, Zoological Institute, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan

Communicated by Richard B. Setlow, December 26, 1990 (received for review October 15, 1990)

ABSTRACT To develop a specific-locus test (SLT) systemfor environmental mutagenesis using vertebrate species otherthan the mouse, we first established a tester stock of the fishmedaka (Oryzias latipes) that is homozygous recessive at threeloci. The phenotypic expression of these loci can be easilyrecognized early in embryonic development by observationthrough the transparent egg membrane. We irradiated wild-type males with '"Cs -rays to determine the dose-responserelationships for dominant lethal and specific-locus mutationsinduced in sperm, spermatids, and spermatogonia. Throughobservation of322,666 loci in control offspring and 374,026 lociin offspring obtained from 0.64-, 4.75-, or 9.50-Gy-irradiatedgametes, specific-locus mutations were phenotypically detectedduring early development. These putative mutations, desig-nated "total mutation," can be recognized only in embryos ofoviparous animais. The developmental fate of these mutantembryos was precisely followed. During subsequent embryonicdevelopment, a large fraction died and thus was unavailable fortest-crossing, which was used to identify "viable mutations."Our medaka SLT system demonstrates that the vast majorityof total mutations is associated with dominant lethal mutations.Thus far only one spontaneous viable mutation has beenobserved, so that all doubling calculations involving this end-point carry a large error. With these reservations, however, weconclude that the quantitative data so far obtained from themedaka SLT are quite comparable to those from the mouseSLT and, hence, indicate the validity of the medaka SLT as apossible nonmammalian test system.

The mouse specific-locus test (SLT), established primarily byW. L. Russell (1, 2), has been used extensively for assess-ment of genetic risks from environmental mutagens. At-tempts to use other species of vertebrates, the guppy (3-5)and the zebrafish (6), for the SLT have been made. However,results obtained were not thorough enough to establish SLTsusing these species. We propose that a fish, the medaka(Oryzias latipes), would provide a valid alternative SLTsystem to study environmental mutagenesis. The medaka isa small freshwater teleost native to Asian countries (Japan,Korea, China, etc.), whose basic biology including classicalgenetics was well established by Yamamoto (7). Further-more, our preliminary data (8-15) and a preliminary report (8)on -irradiation-induced mutations in the male medaka at aspecific locus, b, suggest that the medaka is a promisingcandidate for such a test. In our preliminary study (8), wefound that many color mutant embryos, whose mutant phe-notype could be recognized very early after fertilization, diedduring subsequent embryonic developmental stages but be-fore hatching. Based on these observations, we proposed theterms "total mutations" for specific-locus mutations that

were phenotypically detected during early development and"viable mutations" for hatched viable mutants. It should benoted that the studies on silkworm eggs by Tazima (16) werethe first to show that some egg-color mutants were unable tosurvive to maturity. Herein we report the development of themedaka SLT system, compare the results obtained withinformation accumulated from the mouse SLT system, andthus characterize our medaka SLT system.

MATERIALS AND METHODSMedaka Stocks. The establishment of a multiple recessive

tester stock is a prerequisite for a SLT. Out ofabout 70 genesarising from spontaneous mutations identified in the medakaOryzias latipes (7), we chose the following three autosomalvisible recessive mutations as markers: b (colorless melan-ophores), if (leucophore-free), and gu (guanineless) (17).These color genes were chosen because their phenotypes canbe easily recognized during early embryonic development-i.e., at 2, 3, and 4 days after fertilization for the loci b, if, andgu, respectively. The integration of these three mutant genesinto one individual did not result in reduction of viability andfecundity, which we experienced (11) after establishing ourfirst tester stock containing five marker genes. The testermedaka stock with three markers is currently in its eighthgeneration of brother-sister matings toward development ofan inbred tester strain. A preliminary report on the produc-tion of the tester has appeared elsewhere (17).

-Irradiation. About 0.5- to 1-year-old wild-type malemedaka were obtained from our wild population stocks (stock20) maintained for >6 years as a closed colony in ourlaboratory under the auspices of the Ministry of Education,Science, and Culture, Japan (18). These male medaka inwater at room temperature (-270C) were exposed to y-raysfrom a 111-TBq 137Cs source (Research Center for NuclearScience and Engineering, University ofTokyo) at a dose rateof 0.95 Gy/min. The doses primarily used during the initialphase ofthis study were 4.75 and 9.50Gy; however, the effectof a lower dose, 0.64 Gy, was also examined in later exper-iments. The radiation dose will be given in terms of the rad(1 Gy = 100 rad) when we compare the present results withpublished results using the rad.

Collection and Incubation of Embryos. The experimentalprocedures were essentially the same as described (8).Briefly, in a room (=20 m2) where water temperature andphotoperiod were controlled, respectively, to 27 ± 20C and14-hr light/b-hr dark cycle, each of the irradiated males wasmated with one nonirradiated tester female. A female medakacan lay =20 eggs every day for more than half a year.

Abbreviations: SLT, specific-locus test; DL, dominant lethal; TMF,total mutation frequency; VMF, viable mutation frequency; DD,doubling dose.*To whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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2546 Genetics: Shima and Shimada

Table 1. Number of animals scoredDead SLT

Irradiation Fertilized embryos,Stage dose, Gy eggs, no. no. Total Viable

Sperm 0.64* 4,946 567 12/14,133 2/13,1374.75t 10,970 3,777 144/24,128 16/17,1339.5Ot 8,612 5,013 203/12,838 8/6,099

Spermatids 0.64* 9,201 759 11/26,671 0/25,3264.75t 17,869 4,014 118/36,223 10/30,0849.50t 13,264 4,858 149/19,401 9/10,431

Spermatogonia 0.64*4.75t 71,152 5,431 67/151,547 8/145,2399.50t 43,731 3,481 51/89,085 7/85,413

Control Ot 136,304 8,444 12/322,666 1/304,821Total 316,049 36,344

Maturation stage of male gametes at time of irradiation is shown. Total SLT is (number of total colormutants)/(sum of effective numbers of loci of embryos). Viable SLT is (number of viable colormutants)/(number of hatched fry x number of marker loci per tester used).*Tester stock with three marker loci was used.tTwo tester stocks with one or three marker loci were used.

Fertilized eggs were collected and plated one to a well in96-well plastic microtiter plates, which allowed us to followthe developmental fate of each embryo. The incubation ofembryos in microtiter plates was done under the above-mentioned temperature and light conditions. The maturationstage at which male germ cells were irradiated wasjudged bythe time between irradiation and fertilization as follows (19):fertilization 1-3 days after irradiation corresponds to irradi-ated sperm, 4-9 days after irradiation corresponds to irradi-ated spermatids, and 30 or more days after irradiation cor-responds to irradiated stem spermatogonia.

Scoripg ofPhenotypes. At least once a day, we observed theembryos in microtiter plates under a stereoscopic microscope(Olympus model SZH) for phenotypic expression ofthe threeloci. Daily records of each embryo were made for everyobservable phenomena such as site and relative extent ofpigmentation, eye and trunk morphogenesis, angiogenesis,blood circulation, survival, and hatching.

Calculations ofDominant Lethal (DL) Rates, Total MutationFrequencies (TMFs), and Viable Mutation Frequencies(VMFs). The spontaneous lethal rate of medaka embryos wascalculated in the nonirradiated controls as (number of fertil-ized eggs minus number of viable offspring)/(number offertilized eggs) (8). Then, the induced DL rates were calcu-lated in accordance with Abbotts correction formula, DL =(observed lethal rate minus spontaneous lethal rate)/(1 minusspontaneous lethal rate) (20, 21). To include mutants thatwere phenotypically recognized during early embryonic de-velopment but died before hatching, we defined the'TMFs as(number of total color mutants)/(sum of effective number ofloci per embryo). For example, if the phenotypes of the b andIf loci of an embryo were judged to be wild type and if theembryo died before being judged for the gu locus, then theeffective number of loci for this embryo was scored as 2 (8).The definition for the VMFs, which corresponds to the"mutation frequencies" generally used in animal experi-ments, is VMF = (number of viable color mutants)/(numberof hatched fry x number of marker loci per tester used).

RESULTSNumbers of Scored Animals. The numbers of animals

summarized in Table 1 were accumulated by August 15, 1990.In experiments examining effects of 0.64 Gy, which westarted in July 1990, we have been using the tester medakawith the three marker loci exclusively. However, in experi-ments on dose groups of 0, 4.75, and 9.50 Gy, which were

started in 1986, two kinds of testers, one with one marker andthe other with three marker loci, have been used.Dose-Response Relationship for DLs. Fig. 1 shows DL rates

as a function of y-tray dose delivered to medaka sperm,spermatids, and spermatogonia. In the nonirradiated controlwhere some ofembryonic deaths could be environmental, weobserved that 8444 embryos died out of 136,304 embryos.Thus, the spontaneous lethal rate was 8444/136,304 or 0.0619per gamete (Tables 1 and 2), which was normalized to 0 on theordinate in Fig. 1. Our results for the radiation-induced DLsin the medaka are essentially in accordance with thosereported for the mouse (22) in that no significant radiation-induced increase in DL rates could be found for spermatogo-

0.6Dominant lethals

0.5Sperm

0.4

CZ

03 / Spermatids

0.2

4S ~~~~~~SpermatogoniaI

0) ().64 5 0

Irradiation dose, Gy

FIG. 1. DL rates induced in male gametes ofthe medaka by acutey-irradiation. Wild-type male medaka were exposed in 25'C water to13'CS -rays at 0.95 Gy/min. The irradiated males were mated withnonirradiated tester females carrying three marker loci. Fertilizedeggs were collected and plated in 96-well plastic microtiter plates atone egg per well. The maturation stage of male gametes at irradiationwas assigned by the number of days between irradiation and fertil-ization as follows: 1-3 days after irradiation, sperm; 4-9 days afterirradiation, spermatids; 30 or more days after irradiation, sper-matogonia (19). The spontaneous lethal rate of medaka embryoscalculated in the nonirradiated controls, by (number offertilized eggsminus number of viable offspring)/(number of fertilized eggs) (8),was 0.062. Then, the induced DL rates were calculated by Abbottscorrection formula where DL = (observed lethal rate minus spon-taneous lethal rate)/(1 minus spontaneous lethal rate) (20, 21). Theupper and lower 95% confidence limits were so small that they fellwithin the size of each symbol.

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Table 2. Comparison of estimated spontaneous mutation rates ofmale gametes of the mouse and medaka

Spontaneous rate

Genetic endpoints Mouse Medaka

DLs, no. x 102 per 5* 6.2 (6.3/6.1)tgamete

Total mutations, no. 3.7 (6.5/2.1)tx 105 per locus

Viable mutations, no. 8.10* 3.3 (18.6/0.6)tx 106 per locus

*Data are from Nomura (22).tUpper and lower 95% confidence limits ofthe observed frequencies.*Data from Russell and Kelly (1).

nia, whereas a dose-associated increase over the controlvalues within the dose range examined was found for spermand spermatids.

In Table 3, the estimated DL rates (per rad) are listed withthose for the other two genetic endpoints, for comparison ofthe present data with the mouse data. It should be noted thatdue to the paucity of the data points of the medaka study, noregression analyses were made.By using data shown in Tables 2 and 3, we tried to calculate

the doubling dose (DD) for the increase in DL rates. Table 4summarizes the DDs obtained in the present study for variousgenetic endpoints and spermatogenic stages at the time themedaka was irradiated. Because we had not yet examined theDL rate of spermatogonial cells exposed to 0.64 Gy, thesedata are specified in Table 2 as not determined. As shown inTables 2-4, the results for the medaka are almost the same asthe results for the mouse with regard to spontaneous rate,induced rate (per rad), and DD for sperm.Dose-Response Relationship for Total Mutations. In the

nonirradiated control, we have obtained 12 (6 b, 1 If, and 5 gu)spontaneous total mutations out of322,666 loci examined, fora spontaneous TMF of 3.72 x 10-5 per locus (Tables 1 and2).As shown in Fig. 2, we observed a marked differential

response in terms ofTMFs among the spermatogenic stages.The TMF steadily increased with increasing dose in all threestages. However, it is noteworthy that the TMFs of spermand spermatids are about one order of magnitude higher thanthose for spermatogonia.

Table 4. Comparison of estimated DD for y-ray exposure ofmale gametes of the mouse and medaka

DD, rad

Genetic endpoints Sperm Spermatids SpermatogoniaDLsMouse 64* 37*Medaka 70t, 98t 181t, 170t NDW, 1925t

Total mutationsMouseMedaka 2.9t, 2.9t 6.3t, 5.5t NDt, 44*

Viable mutationsMouse 11.6§ 6.6§ 37¶Medakall 1.4t, 1.7t NDt, 4.7t NDt, 30tND, not determined.

*Data are from Nomura (22); we calculated the data presented bydividing the spontaneous rate by the induced rate (per rad).ISpontaneous rate divided by the induced rate (per rad) for 64 rad.*Spontaneous rate divided by the induced rate (per rad) for 475 rad.§See Table 3, footnote ¶.1Data from Russell and Kelly (1), 8.10 x 10-6/21.9 x 10-8.'Only one spontaneous viable mutation observed in 304,821 loci wastested.

Table 3 summarizes induced TMF (per rad) at 64 rad or 475rad for sperm, spermatids, and spermatogonia. As above, noregression analyses were made due to the paucity of the data.By using the spontaneous TMF and the TMF data at 64(except the point for spermatogonia) and 475 rad, we calcu-lated DDs for the TMF with regard to the stage of the gamete(sperm, spermatids, and spermatogonia) at the time of irra-diation (Table 4). As shown in Fig. 2, although TMFs areabout one order of magnitude higher than the correspondingVMFs [because the spontaneous rates ofthe two frequenciesare also about one order of magnitude apart (Table 2)], theDDs of the two endpoints obtained from the medaka SLTappeared to be almost the same.Dose-Response Relationship for Viable Mutations. The

VMFs also showed a dose-dependent increase, although oneorder of magnitude lower than the TMF (Fig. 2). A markeddifference with a trend similar to the TMF was found againbetween spermatogonia and post-spermatogonial germ cells.The results for the viable mutations can be directly comparedwith the similar results from the mouse.

In the nonirradiated control, only 1 (at the gu locus) of 12spontaneous total mutant embryos survived for test-crossing.

Table 3. Comparison of estimated induced mutation rates per rad for male gametes of the mouse and medakaInduced mutation rate (per rad)

Genetic endpoints Sperm Spermatids SpermatogoniaDLsMouse, no. x 105 per gamete 78* 136*Medaka, no. x 105 per gamete 88t, 63* 34t, 37t NDt, 3.2t

Total mutationsMouseMedaka, no. x 107 per locus 127t, (228/66)§, 125*, 59t, (112/26)§, 68t, NDt, 8.5t, (11.4/6.0)§

(147/105)§ (82/56)§Viable mutationsMouse, no. x 108 per locus 701 1201 21.911Medaka, no. x 108 per locus 233t, (866/36)§, 196t, NDt, 69t, (129/34)§ NDt, 11t, (23/2)§

(320/117)§ND, not determined.

*Data are from Nomura (22).tInduced rate (per rad) for 64 rad.tInduced rate (per rad) for 475 rad.§(ULiiradiated - LLspontaneous)/(LIqnradiated - UIspontaneous) where UL and LL are upper and lower 95% confidence limitsof observed frequencies, respectively.$Data are from W. L. Russell cited by Sega et al. (23). Rates of 2.0 x 10-4 per 300 R (or 285 rad that we calcualted usinga conversion factor of 0.95 rad/R) for sperm and 3.4 x 10-4 per 300 R for spermatids, respectively."Data are from Russell and Kelly (1).

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2548 Genetics: Shima and Shimada

16.3 Ttal

0

Viable

FIG. 2. Frequencies of specific-locus mlltations inducTtal104

00Z Viable

-5 ~~~~~~~~~~~Viable10

0 2 4 6 8 10 0 2 4 6 8 0 2 4 6 8 10

Irradiation dose, Gy

FIG. 2. Frequencies of specific-locus mutations induced by acute

t-irradiation of the male medaka. Frequencies in sperm, spermatids,and spermatogonia were calculated. To include mutants that werephenotypically recognized during early embryonic development butdied before hatching, we used the TMF [(number of total colormutants)/(sum of effective numbers of loci of embryos)] (solidsymbols). The endpoint comparable to "mutation frequencies" gen-erally used in animal experiments is the VMF [(number of viablecolor mutants)/(number of hatched fry x number of marker loci pertester used)] (open symbols). Data are not yet available for thefollowing points: VMF for 0.64-Gy-irradiated spermatids and TMFand VMF for 0.64-Gy-irradiated spermatogonia.

Therefore, the spontaneous VMF of the medaka 0.33 x 10-'per locus (or 1/304,821, the upper and lower 95% confidencelimits are 1.9 x 10-5 and 5.8 x 10-7, respectively) (Table 2)is, although a point estimate, of the same order of magnitudeas the mouse spontaneous VMF (0.81 x 10-5 per locus) (1).However, we are well aware that we have only one sponta-neous viable mutation from our medaka SLT and hence thatall doubling calculations involving this endpoint carry a largeerror. More data are obviously needed.The induced viable mutation rates (per rad), which were

obtained not by regression analyses but by simple division ofinduced rate by radiation dose, for the acute exposure ofspermatogonia are of the same order of magnitude in themouse and medaka (Table 3). The results given in Table 4,which were obtained by dividing the results in Table 2 bythose in Table 3, indicate that the DD for the medakaspermatogonia is almost the same as that for the mouse,although the former has low accuracy because of the paucityof data for spontaneous viable mutations of the medaka.

DISCUSSIONWe have developed a SLT system for radiation mutagenesisusing a fish, the medaka (Oryzias latipes). Despite the factsthat our medaka SLT system is based on only three loci andthat we have observed thus far only one spontaneous viablemutation, the data are comparable with those obtained fromthe mouse SLT and indicate the validity of the medaka SLTsystem. The low inducibility of DLs in spermatogonia, incomparison with that in sperm or spermatids, can be ex-plained by two possibilities: (i) Spermatogonia have a higherrepair capability than post-spermatogonial germ cells, whichare highly differentiated to obtain motility. (ii) The morelikely explanation is that those gonially derived germ cellswith gross chromosomal damages are eliminated at mitosisand meiosis to which the postmeiotically exposed sperm andspermatids are not subjected.The remarkably different stage sensitivity for both TMF

andVMF can be accounted for by the same explanation givenfor the differential spermatogenic DL response. Post-spermatogonial cells of the medaka exposed to, for example,475 rad had considerably higher values for VMF per unit dose(i.e., 69 x 10-8 and 196 x 10-8 per locus per rad, respec-tively, for spermatids and sperm) than did spermatogonia (11x 10-8).The DD method has been extensively used for genetic risk

assessment in humans (24, 25). The experimental data usedto estimate the DD are obtained primarily from the mouseexperiments. As cited in Table 4, some of our medaka dataappear to almost agree with the mouse DD whereas others donot. For the calculation ofthe DD, the precision forTMF datais obviously greater than that for VMF data (Table 1).Nevertheless, the calculated DD for three spermatogenicstages for these two genetic endpoints coincided well witheach other; 1-3 rad for sperm, 5-6 rad for spermatids, and30-40 rad for spermatogonia. Hanash et al. (26) have re-ported that in a human somatic cell system the loci thatsupport genetic variation are more mutable than the mono-morphic loci. The test system we developed depends on theexistence of genetic variation at the loci employed. Thusthese loci may be more sensitive to radiation than most lociand, accordingly, our estimate of the DD may be biaseddownward.The reciprocal relationship between mouse germ-cell mu-

tagenesis and basic genetics has been extensively studied byL. B. Russell (for review, see refs. 27 and 28). There isconsiderable evidence from the mouse SLT that exposure ofmale germ cells to radiation yields high frequencies of mul-tilocus deletions (29, 30). Our medaka SLT system revealedthat a great majority of the total mutants are selectivelyeliminated during embryonic development. This negativeselection can be explained by assuming that the inducedspecific-locus mutations were associated with deletions thatinvolved more than the specific loci (multilocus deletions) orchromosome rearrangements such as unbalanced transloca-tion or hyploids, which were primarily responsible for theDLs. The extensive selection of the total mutants can beexemplified by calculating the VMF/TMF ratios as summa-rized in Table 5. It seems noteworthy that the degree ofselection as revealed by the VMF/TMF ratio is almost the

Table 5. Ratios of VMFs to TMFs in the medaka SLT systemControl 0.64 Gy 4.75 Gy 9.50 Gy

Stage L VMF/TMF DL VMF/TMF DL VMF/TMF DL VMF/TMFSperm (6.2) (8.9) 5.6 17.9 30.1 15.6 55.4 8.3Spermatids 2.2 17.3 10.2 32.4 11.2Spermatogonia 1.5 12.9 1.9 14.3

L, spontaneous lethal rate (number per gamete); DL, increase in DL rate over the control value (number per gamete).Data for L, DL, and VMF/TMF are expressed x 102.

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same among the three spermatogenic stages and doses ex-amined in spite of a great difference in the frequencies of theprimary event (i.e., TMF). Obviously, the induction of amutation at any of the three marker loci alone does not resultin death of the corresponding mutant embryos. Therefore,our results strongly suggest that the induced specific-locusmutations are primarily eliminated by the associated DLs.This point was recently noted by Ehling (24). These resultsraised the following question that should be substantiated byfuture studies: How does a small increase in the DL rate inirradiated spermatogonia result in the large reduction (-1%o)of the TMF down to VMF by an extent similar to thatobserved for the exposed sperm and spermatids with fargreater increments in induced DL rates? To obtain directevidence that induced total mutations are really mutations,we have developed a method to culture somatic cells fromeach of the spontaneous and induced mutant embryos whenthey become moribund, so that DNA and other analyses canbe done. The use of other mutagens with different modes ofaction should be useful. Indeed, our preliminary experimentsusing a potent chemical mutagen, ethylnitrosourea, demon-strated that 15 out of 17 total mutants obtained from gametestreated at the spermatogonial stage became viable mutants,giving VMF/TMF ratios very close to 1 (unpublished data).Also interesting should be the use ofthe mouse supermutagenchlorambucil (31, 32).

We dedicate this article to the memory ofthe late Dr. Nobuo Egami(1925-1989), Professor Emeritus of The University of Tokyo, whostimulated us to start research using the medaka. Our sinceregratitude is extended to Drs. Seymour Abrahamson, Sohei Kondo,Richard B. Setlow, and Avril D. Woodhead for their interest,criticism, and encouragements during the course of our study. Wethank Dr. Hideo Tomita, Nagoya University, Nagoya, Japan, forkindly providing us with the mutant stocks ofthe medaka. The animalcare and breeding was supported by Ms. Shizue Takada and TeruyoTachibana under the auspices of the Ministry of Education, Science,and Culture, Japan. Financial support from grants-in-aid (01602012,01304054, and 01440093) from the Ministry of Education, Science,and Culture, Japan, is greatly appreciated.

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