AfiAiYSIS OF POSflRRADéATI-ON MOIDEHCATION OF GENETEC DM‘AGE {N MATURE DROSQPHILA iviELANéGASYER SPERM 1116355 écr the Degree at? Eh. I). MiCWGAN STA? UNW‘ERSEITY James 3'3. Tmska W63 THESII LIBRARY Michigan State {Indv1n3ity This is to certify that the thesis entitled ANALYSIS OF POSTIRRADIATION MODIFICATION OF GENETIC DAMAGE IN MATURE DROSOPHILA MELANOGASTER SPERM presented by Jams E0 TI’OSkO has been accepted towards fulfillment of the requirements for _PL29_9._ degree in M wan Major Wsor Date August 8; I963 0-169 ABSTRACT ANALYSIS OF POSTIRRADIATION MODIFICATION OF GENETIC DAMAGE IN MATURE DROSOPHILA MELANOGASTER SPERM by James E. Trosko This study was conducted to elucidate the recovery phe- nomenon which is reported to be associated with postirradi- ation modification of the genetic damage in mature Drosophila spermatozoa. In order to distinguish between differential sensitivity or other postirradiation phenomena, the possible modifying effect of several variables was analyzed. Var- iables such as storage of sperm in males and females, cold temperature, physiological condition of the females, and sperm loss were utilized. Sex-linked lethal mutation rate, egg-hatch and counts of total progeny were used to measure the damage in irradiated mature Drosophila spermatozoa. Comparisons of these meas— ured criteria were made between sperm, either non-aged or aged under experimental conditions. The results indicate that neither aging for two weeks nor cold temperature of 10°C changed the sex-linked mutation rate when the irradiated sperm were stored in the females. by James E. Trosko Pre-aging the females on minimal medium at room temperature prior to mating increased the frequency of detected sex- linked lethals. Although lower rates of biological damages were observed in the second sperm batch than in the first sperm batch of all irradiated males, the difference between the two sperm batches was smaller for the males stored at room temperature than non—stored or cold-stored males. The damage associated with the first sperm batch of cold-stored irradiated males was not different from that of the first sperm batch of non— stored irradiated males. These latter two observations were interpreted to indicate that mixing of sperm, rather than recovery, occurred in males stored at room temperature prior to mating. This explanation demands that the sperm popula- tion at the time of irradiation be heterogeneous and compart- mentalized with respect to radiation sensitivity. The cold temperature storage did notsflgnificantly modify the measured damage. However, it was observed after comparing the results of the sex—linked and dominant lethal studies that the cold temperature post—treatment of the irradiated males may have increased the recovery of sex-linked lethals while decreasing the frequency of dominant lethals. ANALYSIS OF POSTIRRADIATION MODIFICATION OF GENETIC DAMAGE IN MATURE DROSOPHILA MELANOGASTER SPERM By IS 6“ ,3 James EéNTrosko A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Zoology 1963 (II-:2 Cf "DNI 5 , ‘31,! lei-“oi ACKNOWLEDGMENTS I wish to thank my wife, Kay, for the technical assist- ance and patience that she afforded me. I acknowledge the fact that the completion of this work was greatly enhanced by the training and guidance from Dr. Armon F. Yanders. To Dr. Philip Clark, I am grateful for his professional advice in the statistical analysis of my data. Also, I wish to single out Dr. G. B. Wilson and Dr. J. Fairley for their contributions to my academic training. I want to thank Dr. U. V. Mostosky of the Department of Surgery and Medicine for the X—ray treatments and also, Robert Wheeler who built the equipment used in the dominant lethal study. This study was conducted during the tenure of a National Defense Education Act Predoctoral Fellowship and partially supported by a grant to Dr. A. F. Yanders from the U. S. Atomic Energy Commission (Contract AT (ll-l) - 1033). ii Acknowle List of List of SECTION I. II. III. IV. V. APPENDIX TABLE dgments . . . . . Tables . . . . . . Figures . . . . . INTRODUCTION . . . OF CONTENTS MATERIALS AND METHODS . . . . RESULTS . . . . . Stored Females Stored Males DISCUSSION . . . . Stored Females Stored Males SUMMARY . . . . . BIBLIOGRAPHY . . . . . . . iii Page ii iv 13 23 23 29 35 38 45 Table 1. II. III. IV. VI. VII. VIII. IX. LIST OF TABLES Page Experiment I. Comparison of the number of radiation-induced sex-linked lethal mutations in non-stored spermatozoa and in spermatozoa cold stored for fourteen days . . . . . . . . . . . . . . . . . . . 9 Experiment II. Results of cold storing irradiated sperm in cold-prestored females 10 Experiment III. Effect of non-storage and storage of control and irradiated sperm on the average total progeny from pretreated females . . . . . . . . . . . . . . . . . . ll Experiment IV. Results of storing males after irradiation on the recovery of sex- linked lethal mutations . . . . . . . . . . 16 Statistical results from experiment IV . . 18 Experiment V. Results of storing males after irradiation on the recovery of dominant lethals . . . . . . . . . . . . . l9 Experiment II. Detailed results of cold- storing irradiated sperm in cold prestored females 0 O O O O O O O O O O O O O O O O O 39 Experiment IV. Individual records of sex- linked recessive lethals from NS, SRT, SCT— irradiated males . . . . . . . . . . . . . 40 Experiment V. Individual records of dominant lethals from control, NS, SRT and SCT—irradiated males . . . . . . . . . . . 42 Results of t tests on dominant lethal data of experiment V . . . . . . . . . . . . . . 44 iv Figure LIST OF FIGURES Page Experiment IV. Results of storing males after irradiation on the recovery of sex— linked lethal mutations . . . . . . . . . . . 17 Experiment V. Results of storing males after irradiation on the recovery of dominant lethals O O O O O I O O O O O O O O O O O O O 20 I. INTRODUCTION In order to pursue a study on possible postirradiation modification in mature Drosophila sperm, those factors which might influence measurement of the damage must be clearly delineated. Stated in such studies are the concepts of re- covery and differential sensitivity in mature sperm (Baker and Von Halle, 1953; Bonnier and Lfining, 1953; Bonnier, 1954; Lfining and Hannerz, 1957; Lfining, 1961; Oster, 1961). The recovery concept has been based on the observations of higher mutation frequencies associated with sperm utilized on the first day after irradiation rather than with sperm utilized on the second day, whether or not the male was mated on the first day (Baker and Von Halle, 1953; Lhning, 1954; Telfer and Abrahamson, 1954; Nordback and Auerbach, 1956; Yanders, 1959a; Oster, 1961; Alexander, 1962; Kvelland, 1962; Reddi and Mathew, 1963). Some investigators, however, have not always observed this phenomenon (Oster, 1959; Alexander and Bergendahl, 1962). Alternatively, the observed difference in biological damage associated with the first and second sperm batches may be due to differential sensitivity. Differential sen- sitivity, possible due to oxygen gradient, chemical, protec- tion or systems of metastable energy are implicated by the works of Bonnier (1954), Auerbach (1954), Nordback and Auer- bach (1956), Lfining (1954 and 1958), Khishin (1955), Yanders (1956), Oster (1959), Stromnaes (1959) and Wolff and Lindsley (1960). There may be other postirradiation phenomena which could modify the measured damage in mature sperm with or without a recovery process (Clark, 1960). Some investigators have presented evidence that postirradiation enhancement of the damage is possible in systems where recovery processes have been shown not to occur. Novitski (1949) found that a cold shock post-treatment given to irradiated inseminated fe- males increased the mutation frequency. Since it has been demonstrated by Novitski (1947), Abrahamson and Telfer (1956), Lee (1958) and Oster (1961) that no recovery of sex- linked or dominant lethals occurs after aging irradiated sperm in the female, this increase Would not be due to an inhibition of a recovery process. Alexander (1962) showed that when mature sperm are treated in inseminated females with X rays in the presence of nitrogen gas, there is a postirradiation enhancement of radiation damage. Herskowitz (1958 and 1963) has shown that undernourishment of the irradiated, inseminated females will lead to an increased amount of chromosomal aberrations when compared to well nourished females. The experiments undertaken in this study were conducted to analyze some postirradiation variables which might in- fluence the measurement of the radiation damage. Variables such as storage of sperm in males and females, cold tem- peratures, physiological condition of the females, and sperm loss were studied in reference to postirradiation modification. To eliminate any indirect effect of the radiation on the females, all mature sperm were irradiated in the males. To test whether or not recovery or enhancement of radiation- induced damage in the mature sperm occurred in the female, a comparison was made between non—stored and stored inseminated females. Since storage of inseminated females at room temperature indicated no recovery of the radiation-induced damage in the sperm (Novitski, 1949), any modification after storage at cold temperatures would suggest mechanisms of postirradiation enhancement of the damage. To test between recovery or differential sensitivity, experiments were designed to store males after irradiation at room and cold temperatures. If a recovery process oc- curred at room temperature, the stored males would be ex— pected to have a lower mutation frequency associated with their sperm than non-stored males. Since the males stored at room temperature would be expected to be continuously producing more sperm, conceivably this new mature sperm might mix with the older sperm, hence modifying the measured damage. To distinguish between recovery and mixing, males were stored at 10°C after irradiation. The cold temperature might be expected to interfere with an active metabolic re- covery mechanism. At the same time, it might inhibit devel- opment of any more new mature sperm. After the cold storage of the male, one should expect very little difference in the induced biological damage when compared to the non-stored males, assuming there is no postirradiation enhancement due to the cold temperature. Sex-linked mutation rates, egg-hatch and progeny numbers were studied. Comparisons of these criteria are made between the non-stored males or females and the experimentally stored flies. II. MATERIALS AND METHODS Stocks of Drosophila melanogaster, Oregon-R (OR), wild- type strain, and Ba§g_X—chromosome tester strain (Muller-5) were used exclusively. In all experiments, virgin OR males were aged three days on nutrient medium (modified after Carpenter, 1950) prior to treatment. Males were exposed to 0 (control) or 2760 r units of X rays in each experiment. Radiation was administered with a General Electric Maximar-250-III, operating at 250 kv, 15 ma, with a 50 mm copper filter, giving an average dose rate of 150 r/minute. For the sex-linked lethal experiments in which the females were stored after insemination, virgin Muller-5 females were stored on Offerman's syrup-agar medium (1936) for seven days prior to mating, either at room temperature (experiment I) or at 10°C (experiment II). This was done to eliminate egg-laying during the mating and subsequent storage period (Trosko and Myszewski, 1962). In experiment IV, where some males were stored after irradiation, and in experiment V, virgin Muller-5 females were aged two days on a nutrient medium prior to mating. Flies were mass—mated on Offerman's medium at room tem- perature for experiments I and II. After twenty-four hours, males were discarded and the females were divided into two groups. One group was placed on nutrient medium (twenty- five females per bottle) and transferred every two days until all sperm were depleted. The other group was stored at 10°C on Offerman's medium for two or more weeks. At the end of this storage period, they were placed on nutrient medium and treated in the same way as the non-stored group. All F1 fe- males were tested for recessive sex-linked lethals (Auerbach, 1962). In experiment III, virgin Muller—§_fema1es were collected within eight hours of eclosion. One group of these females was mated immediately to three-day old 0R males and either allowed to lay eggs immediately or stored on Offerman's min— imal medium for two weeks. The second group was stored one week prior to mating on either minimal medium or nutrient medium. After mating these latter females, they were allowed to either lay eggs in individual vials or stored for two weeks on minimal medium before being allowed to lay eggs. Once the females were allowed to lay eggs, they were trans- ferred every two days until all sperm were depleted. A11 progeny were counted. The experiment was run in duplicate, with one set utilizing non-irradiated OR males, the other with OR males irradiated with 2760 r units. In experiments IV and V, the male flies were individ- ually mated. One Oregon-R male was mated to three virgin Muller-§_fema1es for twenty-four hours (sperm batch 1) and then transferred to a second set of three virgin Mullerfii females (sperm batch 2). After the second mating of twenty- four hours, the males were discarded. In the sex-linked lethal mutation experiments, the females were then trans- ferred to new vials every two days until all sperm were depleted. All F1 females were tested for sex-linked lethals. In the dominant lethal studies, the mated Muller-§_females were placed individually in vials which were inverted on a petri dish of nutrient medium (charcoal was added to facil- itate counting) for twenty-four hours and transferred once to another petri dish for an additional twenty-four hours. Egg hatch was checked thirty to thirty-four hours after re— moval of the females from the medium. In experiments IV and V, the males that were held at 10°C were so subjected within ten minutes after irradiation and were mated immediately upon removal from the cold tem- perature after the twenty-four hour storage period. III. RESULTS Stored females The results of scoring sex-linked lethals for cold- stored females are summarized in Tables I and II. To test for possible postirradiation modification of the genetic damage, the percentages of sex-linked lethals induced in the mature sperm contained in the non-stored females are compared to those of the stored females. In experiment I, in which the Muller-§_females were prestored at room temperature on Offer- man's medium prior to mating, there was always an increase in recoverable lethals following storage after irradiation. Although none of the separate experiments showed a statis- tically significant increase of lethals after storage, a chi- square value of 19.37, with ten degrees of freedom (0.05 > P > .025), is obtained when Fisher's method of com- bining separate probabilities is employed (Fisher, 1950). The combined results indicate that there was a significant increase in sex—linked lethals after storage. When the Muller-§_fema1es were prestored at 10°C prior to mating (Table II), there was no apparent change in the lethal rate after two weeks of storage. A chi-square value of 0.11, with two degrees of freedom, was obtained when oooa :msu umsumu oOmH um ommuoum mcsu woman no muHSmmH cmaoom a N AEOGmem mo common 020v mummy hocwmsfiucou mumsvnlflfio Nxm mo muHSmmm Ass mnsumummfiwu Eoou um mcflume on Howum ammo cm>mm Eswcma HmEHsHE so pmuoummum mmamamm es :oflumfiemunfl mo mafia: A comm A uuuuuuuuuuuuu u--- mm.o mmmH\a m~.o mmma\a Houucoo mmo. A m A mo. no.a mm.oH HHoH\m6H ev.m ommm\mmm m cm. A m A ma. mH.o mH.m onma\mva am.m mmhm\mmm No mo. A a A oa. am.m mm.oa mam\ao mm.m omoa\am 6 mo. A A A OH. on.m mm.HH amm\nm mm.m mooaxam m oa. A m A mm. mm.H mm.HH : .. oma\ea ma.m Hmmmxemm Ha A N Hanson s. Hmerzmemnumu Hagumu a. HmerZ\mHmsumu umm «Ra. Umuoum Umuoumlcoz ssmmmp smmuusom Mom Umnouw taco monoumfihmmm CH cam moNoumEmem Umnoumlcoc ca msoflumusfi Hmnuma pmxcfialxmm epmosccHIGOHDMAUmu mo Hones: can mo somHHmmEoo .H usmeflnmmxm .H canoe 10 Results of cold storing irradiated* Table 11. Experiment II. sperm in cold-prestored females** Set Letha1s***/Normal Length of Storage % Lethal F 143/1733 0 days 7.62 G 213/2508 1 day 7.83 H 239/2795 12 days 7.88 * 2760 r units of irradiation ** Females prestored on minimal medium at 10°C seven days prior to mating *** Sex-linked lethals 11 oooa um Esflme m.cmEHmmmo so mhmo sm>om How .mcflumE ou Hofium .pmuoummum u D mnsumummfimu Boon um Eswomfi m.cmEmemo so mmmc cm>om Mom .mcflumfi ou HOAHQ .pmhoummum n B mnsumummamu Eoou um finance ucwfluusc so mmmp cm>mm How .mcfiumfi on Hofium .Umhoumwum u m Unaumfi cu HOHHQ mmHmEmm UHOIHDOQIHDOMI>DGm3qu u m muses A omnm spas omumflemuufl euodm u + wuomwumo >Hm>m CH mmHmEmm sou mo mammoum mmmum>m usmmmnmmu mama u A me OOH NHH Ohm mm D mm mm on Hma am 9 mm nma mHH mud do w mm med mm Hmm mm m +Eumam poumwpmuuH Emmmm Houusou +Euwmm pmumapmnuH Enemm Houusoo pom Umuoum wwHOHmlcoz «mmamfiom cmumwnumum Eoum wcmmOHm Hmuou mmmuw>m ma» :0 Enwmm UduMHpmun cam Honucoo mo mmmuoum cam mmmnoumlco: mo uummmm .HHH ucmEflHmmxm .HHH OHQMB 12 testing for heterogeneity between the non-stored groups compared to the one-day and fourteen-day stored groups. Storage at 10°C had a drastic effect on the total progeny output of the Muller-§_fema1es, depending on the pretreatment of the females prior to mating. In experiment I, the cold- stored females gave very few progeny. Dissection of some of these females immediately after two weeks of storage showed an ample number of sperm in the storage organ. Obviously, these stored, inseminated females lost sperm sometime after removal from the cold temperature and before egg-laying resumed. No significant reduction in progeny numbers was observed when the Muller-5 females were prestored at 10°C immediately after eclosion prior to mating and storage. Experiment III (Table III) indicates the effect of dif- ferent pretreatments on the fecundity of the female. It is seen from these data that prestoring the female at room temperature on minimal medium for seven days prior to mating decreases the total progeny number whether or not the female is subsequently stored after mating. This suggests why low numbers of offspring were obtained in experiment I. The fe- males of set u, treated as in experiment II, gave more progeny than did those treated as in experiment I (set T). However, a drop in progeny number after storage of set u females was 13 not observed in experiment II. Some other factor, possibly non-constant humidity, may have caused this discrepancy. Stored males Tables IV and VIII summarize the percentages of radia- tion—induced sex-linked lethals of postirradiation-stored males. In the non-stored (NS) and cold-stored (SCT) groups, the second—day sperm batch had a lower mutation frequency than the first-day sperm batch. This was not true for the males stored twenty-four hours at room temperature prior to mating. A chi—square test for heterogeneity between the non-stored (NS), room temperature stored (SRT) and cold- stored (SCT) males gave a value of 1.63, with two degrees of freedom (0.50 > P > .25). A chi-square test comparing the NS-males with the SCT-males gave a value of 1.56, with one degree of freedom (0.25 > P > .10). The results of these latter two chi-square tests indicate that the manner of post- treatment does not change the frequency of recovered sex- linked lethals. In order to analyze variation among individual NS-males and SCT—males, a t test was made on the total results of the two days of mating. A t value of 0.99, with nineteen degrees of freedom (.20 > P > .15), was obtained. This 14 indicates, as did the results of the chi-square tests, that cold treatment did not change the frequency of recovered lethals. Also, t tests were used in comparing the first and second sperm batches of NS-males and SGT-males. A Student's t value of 0.17, with nineteen degrees of freedom (.45 > P > .40), was obtained for the comparison between the first-day sperm batches of the two groups. This suggests that no recovery occurred in the SCT-males' sperm, and that the cold temperature did not enhance the damage. The meas- ured damage associated with the first sperm batch of the SCT-males was not different from that of the first sperm batch of the NS-males. When the second-day sperm batches of NS and SCT-males were compared, a t value of 1.39, with nineteen degrees of freedom (.10 > P > .05), was obtained. This might indicate that the cold temperature enhanced the damage in the sperm which was used on the second day of mating. Another observation is that the total rate for the first and second sperm batches of the SRT-males was not signif- icantly different from the NS-males' first and second batches. This is indicated by the t value which was computed to be 0.30, with sixteen degrees of freedom (.40 > P > .35). This suggests that there was a mixing of sperm rather than a re- covery in SRT-males' sperm. 15 A similar experiment utilizing dominant lethals is sum- marized in Tables VI and IX. Superficially, it appears that after irradiation the SCT-males had fewer dominant lethals associated with their first and second-day sperm batches than did those which were non-stored or stored at room temperature. Several t tests comparing the different sperm batches are summarized in Table X. On comparing the total egg-hatches of the NS-males and SGT-males, a t value of 0.67, with seventeen degrees of freedom (.30 > P > .25), was obtained. This indicates that the cold temperature did not significantly modify the radiation-induced damage. Al- though the effect of the cold temperature was not statis- tically significant, it was observed that the sex-linked lethal rate was higher for the SCT-males than the NS-males, while the dominant lethal rate was lower for the SCT-males than for the NS-males. The most significant observation of the dominant lethal study was that SRT-males gave an identical egg-hatch to that of the NS-males. This observation was based on the sum of the first and second-day sperm batches. Although the SRT- males' first-day sperm batch showed less damage, as had been shown by Baker and Von Halle (1953), Nordback and Auerbach (1956), Yanders (1959a) and Mathew and Reddi (1963), the 16 Oooa um .mcaumE ou Hoflum .coflumapmnnfl Hmumm mnson HSOMImusm3u monoum mean: u Bow wusumummfimu soon um .mcwume ou Hoflum .GOADMHUMHHH Houmm mnson unauthucmzu wwuoum mean: u amm mmHmE wououmlcoz u wz HHH> magma mo muasmmu census 4 mo.o na.m ems\oa mm.n amHH\mm eom om.m mo.m Hom\om no.6 mmm\am emm mm.m oo.m mmn\nm om.n Hmm\mo mz As amuoe Hmnumq.x mamsuoz\mamsumu Hmzuwu x. mamsqu\mHmsumu pom noumm enema ecoumm goumm enumm umnflm mc0fiumuse nguma poxsflalxmm mo >H0>oowu ms» so GOADMHpmuufl Hmumm mmHmE msfluoum mo.emuasmmm .>H ucmEHHmmxm .>H magma l7 E-I U U) E4 Di U) U) Z «MM w 11W . "Io; . xi ,. I 1.1... m. ..2. , m... my. ,0... , H 1.). ..\ .MK .-- IIHHL T\ an”. “H .1 tnIHJ. . V Isl.» \ .3 «WHO... 4: ... A AKIN”: CIHWU I . .1 ,I . ax. thAn rm 5.1.; ”w J“) . “VIA. .fiVMHI Emu. L as... “Human afv . drum: HAHN MW Hum Aw” I II ALI; ‘fluulfl WM... 1... T... m... «HI, an... . to» s Jtv oooH um omnoum moans neom mm, vim Aw“ mnsumummfimu mm WY WW: fIH/J. .JWFI n\ .1 Eoou um pmuoum moan: “8mm WM WW H: .Hu. W. -,. . AI. mmame cwnoumlcoz umz .Wm nmfl mm .M I. T... ‘1 . v) .31. £0qu Emmmm cam flu Sousa Euwmm uma mm D I ALI}! {1.5} . . V‘ I‘7 . ' .4. Ag , V ‘1‘.”‘0‘1 .ms0flumuse Hmzuwa pmxcaalxwm mo mnw>oomu can so soflumfipmuufl Hmumm mmHmE mcflnoum mo muasmmm .>H ucmfiflnmmxm H musmwm .005 1 SRT lSt vs. 2nd Sperm batch 0.11 P > .50 1 SCT lSt vs. 2nd Sperm batch 4.49 P > .025 1 NS-T vs. SRT-T vs. SCT-T 1.63 P < .50 2 NS-T vs. SCT-T 1.56 P < .25 1 Student‘s t tests T P D. F. st st " I NS 1 vs. SRT 1 Sperm batch 1.12 .15 > P > .10 16 NS ISt vs. SCT lst Sperm batch 0.17 .45 > P > .40 19 NS 2nd vs. SRT 2nd Sperm batch 1.293 .15 > P > .10 16 NS 2nd vs. SCT 2nd Sperm batch 1.39 .10 > P > .05 19 NS-T vs. SRT-T 0.30 .40 > P > .35 16 NS-T vs. SCT-T 0.99 .20 > P > .15 19 NS = Non-stored males . SRT = Males stored at room temperature SCT = Males stored at cold temperature T = Total sperm scored (lSt and 2nd sperm batch combined) l9 mcfiumfi mo who vacuum Eoum Euwmm msfluma mo hop umuwm Eoum Ehmmm m soups ascem H seems sucem Q can 0 upon Mom venous; mmmm Hmuoe n B mcfluma Hmumm muse; unmawlmuuom Esacme ucmummmwp so mmHmem each an pouwmommp mmmm u A mcflumfi Hmumm mason unculmucwsu moa~Emm an pwuflmommc mmmm u m pwsuumnss pom pmzoum£\ponoumm st XH manna mo muasmmu pmmasq s Ruhmm Swab Rico Romém Rimm Xm.¢m Romdm mmmH\Hmm nmc\cmm Hcm\mo~ mmm\HmH Hae\m~c mom\bam mem\mcH concepmnufi pwnoumlpaov dem Xo.mm dem $5.0m Ax.m.~m ANemdm $.me emam\m¢ma moea\mmm mmoa\mmm mmv\wmm mah\hhm om¢\mam mmm\mma beneficmunfl cmnoum dem Redo Xvioo Rats $.me finkme $0.4m mmHH\omo hmm\omm onm\mmm nmm\>ma omm\mmm mmm\hwa o>~\mma poumwpmunfl cmnoumlcoz Rm .om Rm .om RH 13 dem Km 220. RN .mm A3mm meoa\n¢m m¢m\mmm mmm\bmm mmm\mmm mmm\mwm th\hmH mmm\moN Honusoo a A m A m AN+HV m soumm m nuumm m soumm e spasm H soumm a noumm Hmuoe Ehmmm Eummm Summm Enemm Ehmmm Ehmmm «emamguwa uamcflfioc mo hnm>oumu on» so GOHDMAUMHHH Hmumm mmHmE msfihoum mo smuasmmm .> ucmfifihwmxm .H> manna 20 Houucoo 003 em 68806 means ”90m m. wusumumewu J Econ um bemoan mmamz namm .g. mmHmE monoumlsoz umz WV Son—m9 Eummm paw D Luann Ehwmm and mm .mamnuwa HGMCHEOU mo >Hm>oomu 93 so soflumfiemunfi Hmumm memE mcfiuoum mo muasmwm .> ucmfifinmmxm N musmam ION toe seems .x. :00 cm OOH 21 second-day sperm batch showed more damage than the second— day non-stored sperm batch. It appears as if the damage of the total two-day SRT-sperm batch is an average of the NS—sperm batch. A t value comparing the total two-day sperm batches of the NS-males and SRT-males was 0.48, with eighteen degrees of freedom (.35 > P > .30). This shows that there is no significant difference between the total two-day sperm samples of the two types of males. Individual variation of the different males showed up in the total progeny sired. However, the Student's t test took into account these variations. Lfining (1952b, 1957) and Stromnaes and Kvelland (1962) demonstrated that there! is great individual variability. Although the male seems to be capable of inseminating at least three females a day, some only copulated with one or two. Wedvik (1962) showed that temperatures of about 7°C did not affect the male fertility. In summary, postirradiation treatment with cold storage did not have a statistically significant influence on chang— ing the mutation frequency. There seemed, however, to be a parallelism between higher egg-hatch and higher frequency of sex-linked lethals scored in the two sperm batches of the SCT-males when compared to those of NS-males. The average 22 biological damage for the SRT-males seemed to indicate no recovery but rather a mixing of sperm when compared to the NS-males. This suggests that the sperm population at the time of irradiation is heterogeneous with regard to initial radiation sensitivity and that it is compartmentalized with respect to sensitivity. IV. DISCUSSION Stored females The observed higher frequency of sex-linked lethals after storage of inseminated females might be the result of several possible postirradiation phenomena. It was initially thought that the cold temperature was associated with a higher frequency of lethals. Novitski (1949) found that postirradiation cold shocks to inseminated females resulted in higher lethal mutation rates than when post- treated at 25°C. Since it has been suggested by Muller (1940) and Oster (1961) that no recovery occurs in the fe- male, the cold temperature would not be expected to in- hibit a recovery process, but it might have resulted in more lethals due to increased oxygen absorption and inter- action with long-lived products of the irradiation. It was also noted that the increase in lethals was associated with a significant loss of progeny upon storage. This loss was not checked with reference to a dominant lethal study on the eggs of the stored females since a dominant lethal study on these females would have been unsatisfactory be- cause of their low fertility. Although the fecundity of these females was reduced after storage, they still laid 23 24 many unfertilized eggs so it would have been nearly impos- sible to determine the dominant lethal rate unless a cy- tological examination of each egg was made. Lee (1957) has shown that the dominant lethal rate in the honey bee did not change after a year of storage. Of course, it must be kept in mind that the conditions of storage were dif- ferent in my experiments in that I had to starve the Drosophila female to inhibit egg-laying during storage, while this is not necessary for the honey bee. Experiment II, demonstrated in Table II, has definitely shown that the cold temperature treatment p§£_§g_was not responsible for the increase in sex-linked lethals, nor was the twoaweek aging period. There was essentially no loss of progeny during the storage of these females (Table VII). The difference between the results of experiment 1 and 11 seemed to be related to the number of irradiated sperm that survived the storage and took part in post-storage fertilization. The pretreating prior to mating seemed to be correlated with the total number of progeny after the post-storage period was over (Table 111). Hence, it is likely that the higher mutation frequency after storage is related to the condition of the female at the time of storage. 25 In experiment I, the females were very emaciated after a week of storage at room temperature on Offerman's minimal medium. It seems likely that this condition affected the total offspring for both the non-stored and stored females (experiment III, Table III). The actual mechanism for this reduction is not known, but it may be related to exogenous materials from the cells of the female storage organs which may be normally supplied to maintain the sperm. There is evidence that the environment of the sperm might affect the mutability of the paternal chromosomes. Herskowitz (1958) observed a higher translocation rate after females were stored on a minimal medium. Later (1963), he showed that the physiological condition of the female will influence the gross chromosOmal mutational frequency scored from x-rayed sperm. Hildreth and Carson (1957) showed that the frequency of spontaneous lethals in the sperm of wild- type Drosophila melanogaster was influenced by the type of female that the males inseminated. They suggested that maternal influence might exert a differential mutagenic action on the X chromosomes of the sperm during the storage period, or that the different conditions of the females might have a differential influence on the "healing" of lethals that originated in the sperm before mating. The 26 latter might be more applicable to the interpretation of my findings. Bonnier and Lfining (1951) showed that when the eggs were irradiated prior to fertilization, an increase in the duration of time from irradiation to fertilization in- creased the rate of gynandromorphs. Bonnier (1954) found that by irradiating eggs there was an increase in the fre- quency of chromosomal aberrations scored in the X chromo- somes of the sperm. Alexander (1962) indicated that post- irradiation enhancement of radiation damage was possible when sperm were treated in the inseminated females with X rays in the presence of nitrogen. If during starvation or irradiation of the female some eggs are affected irreversibly, repair or "healing" of the damaged chromosomes might be affected. This assumes that "healing" is normally dependent on the plasm of the egg. It may be that abnormal healing will ensue and hence lead to detectable lethals. Of course, whether lethals are the re- sult of rejoining of chromosome breaks has not been satis— factorily shown (Lea and Catcheside, 1945; Herskowitz, 1946; Lfining, Schuwert and Jonsson, 1958). My results and those of Herskowitz would tend to suggest that lethals may result from abnormal rejoining of breaks. Also, with re- ference to starvation of the females, it has been shown 27 that dehydration had no effect on egg mortality (Reitan and t al, (1932) have shown that Annan, 1962). Patterson, after two or three days aged females were normal with regard to egg fertility when compared to non-aged females. There- fore, it seems that undernourishment of the females is only effective when broken chromosomes are present. Abrahamson and Telfer (1956) showed that radiation— induced damage incurred in sperm after insemination was in- dependent of: (1) aging versus non-aging’fl1the females be- fore irradiation; (2) aging versus non—aging in females after irradiation, and (3) aging versus non-aging of the males before they discharged sperm that were to be irra- diated in the females. Since the sperm used in my experi- ments were always irradiated in the male, there might be a factor related to possible sperm competition during sperm transfer and storage in the female. Yanders (1959b) has shown that irradiated sperm behave less efficiently than non-irradiated sperm with reference to successful inseminations. Also, Lindsley, Edington and Von Halle (1958) have reported that there is a relationship between the genetic constitution and the sperm sensitivity to X irradia- tion. Frydenberg and Sick (1960) have deseminated females by cold—shock treatments and found that the efficiency of 28 desemination is largely the property of the sperm. Since these are demonstrations of inter-strain or inter—genetic properties of the sperm's ability to survive, the question of a difference existing in a population of mature sperm within a given male arises. It is obvious from the data on progeny numbers (Table III) and from observations of the dissections of some fe- males that not all sperm participate in successful fertili- zation. There might be a relationship to the sperm that are stored and utilized and those which are lost. Lefevre and Jonsson (1963) have indicated a preferential displace- ment of one type of sperm over another in two sequential matings, and they demonstrated that the overall productivity of remated females did not significantly exceed that of non-remated females. This might suggest that there is a factor related to a sperm's ability to compete for storage. The sperm that are present at the moment of sperm trans- fer far exceed those which can be stored (Kaufmann, 1942; Lefevre, §E_§1,, 1962). It might be that among these sperm is a mixture of sperm which had different sensitivity to the radiation. Kimball (personal communication) suggested that possibly the sperm most capable of surviving transfer 29 and storage are those which are most susceptible to irrad- iation while in the male. Stored males As mentioned in the introduction, the decrease in the radiation-induced biological damage associated with a second- day sperm batch has been demonstrated many times. The de- crease has been argued to be due either to a recovery mech- anism, to differential sensitivity of mature sperm during irradiation, or to a loss of sperm in the stored irradiated males. The evidence of a recovery mechanism depends greatly on the observation that irradiated males, stored one day prior to mating, showed a reduction in biological damage in their first-day sperm batch when compared to the first- day sperm batch of non-stored males. However, it has not previously been shown what degree of damage is present in the second-day sperm batch of the stored male. My results which include this infdrmation (Tables IV and VI) indicate that there is no recovery of the genetic damage in the mature sperm but rather a mixing of sperm having differential sensitivity to the radiation. There is no difference between first and second-day batches when the male is irradiated in a nitrogen atmosphere 30 (Lfining, 1954; Lfining and Soderstrom, 1957), and this sug- gests that anoxia of the second-day sperm batch gives rise to the difference between the two sperm batches. In addi- tion, Alexander's finding (1962) that there is an absence of a difference in the first and second-day tests in sperm irradiated with neutrons suggests that there is no recov- ery. Since a differential oxygen gradient in the testis would not be expected to influence the radiation damage due to neutrons, these results indicate that no recovery process exists, but rather a differential gradient of sperm sensi- tivity in the testis. Because the total damage for the first and second-day sperm batch is lower in a nitrogen atmosphere than in air, LUning has suggested that this is due to the loss of inhibition of the rejoining mechanism. He thinks there is the same amount of chromosome breakage in a nitrogen atmosphere as in air. Wolff (1959), however, has stated that breakage is affected as well as the rejoining mech- anism in air, hence implying less breakage in a nitrogen atmosphere. Muller (1940) has shown that at least some sperm chromo- somes, broken while being irradiated within inseminated fe- males, do not rejoin until after fertilization. He also has stated that the great majority of chromosomes broken by 31 X rays reunite in their original order (in Hollaender, 1953) but, as Oster (1961) points out, this non-specific condi- tioned rejoining does not qualify as a recovery mechanism. The question is: does post—storing of males contribute to an active recovery by supplementing the non-specific re- joining that occurs? This active recovery would have to depend on specific materials and conditions provided by the male. Does the cold temperature enhance the non-specific rejoining rather than inhibit a specific recovery mechanism? My data seem to indicate that at room-temperature storage there is no more rejoining of chromosomes than takes place normally in the non—specific manner described by Muller. At cold temperatures, however, there seems to be an enhancement of this non-specific rejoing, primarily leading to restitution. Since there is no evidence for any substantial sperm loss in the stored male (Muller, 1951; Lfining, 1952; Bateman, 1954), the average of the two-day sampling of damage in the sperm of the SRT-males will essentially include the same sperm sampled in the NS-males. The SRT-males will be continually producing more mature sperm and they would accumulate and probably cause the older, mature sperm to mix (Bateman, 1956; Mossige, 1955). There may be little mixing of the cold- stored sperm because the SCT-males would not be producing 32 many more sperm, if any at all. The cold temperature would have been expected to inhibit a recovery mechanism to some extent. This would have resulted in a small or no difference between the first and second sperm batches, assuming that there is no postirradiation enhancement by the cold tempera- ture. The fact that the difference in sex—linked lethals between sperm batches I and II in the SCT-group had a chi- square value of 4.49 (0.05 > P > .01), suggests that there is actually acifferential sensitivity in the two sperm batches and that there is no recovery from the genetic damage. Yanders (1959b) noted a reduction in the degree of suc- cessful inseminations after irradiation and a "recovery" if the male was held for a twenty-four hour period before mating. If mixing occurred, this "recovery" might reflect a mixing of the older sperm.which are most senSitive ‘bo radiation,with the younger, less sensitive sperm. If the degree of insem— ination actually parallels the same degree of genetic damage in the sperm, one should note less "recovery" of successful inseminations in the second—day sperm batch of SCT-males. An increase in sex-linked lethals and a decrease in dominant lethals appeared in the total sperm batches I and II for the SCT—males over the NS-males, although in neither 33 case was it statistically significant (Tables V and VIII). If this increase is real, possibly the decay of a radia— tion product demonstrated by Lfining and Henriksson (1959) and Wolff and Lindsley (1960) is affected by the cold temperature. To explain the increase in sex-linked lethals and de- crease in dominant lethals of the SCT-males, assuming these differences are real, the cold temperature may have de— creased the number of chromosome aberrations which would lead to dominant lethals. Among those chromosomes "saved" from dominant lethals, a portion would be expected to con- tain sex-linked recessive lethals. In other words, some chromosomes that have recessive lethals induced in them may never be scored because these same chromosomes might have participated in some chromosomal aberration which lead to a dominant lethal. The cold temperature might be expected to decrease the number of chromosome fragment interchanges and increase the chance of restitution of the nature de- scribed by Muller (in Hollaender, 1953). This would increase the amount of sex—linked lethals recovered. It is not known whether an increase of oxygen in the sperm cell occurring during the cold storage could contribute to enhancing genetic damage by reacting with any long-lived 34 radiation products. Novitski (1949) found that when sperm irradiated in females were post-treated at -5°C, there resulted a higher lethal rate than when exposed at 25°C. Because a restoration mechanism has been shown not to exist in the female, the effect of the cold cannot be to inhibit recovery. Instead, it seems that the cold temperature en- hanced the damage. It is concluded from these experiments that, after ir- radiation of the male, the degree of damage produced in the mature sperm is constant depending on the age and location of the sperm in the testis. If the male is not mated, some mixing of the sperm occurs at normal temperatures, but if the male is post-stored at 100C, there is little mixing. Furthermore, enhancement of the damage may occur through restricting interchromosomal exchanges or possibly by in- creased radiochemical activity due to an increase in dis- solved oxygen at cold temperatures. After sperm are trans- ferred to the female, the damage remains constant (no recovery) unless the female is undernourished, which acts to enhance the damage. V. SUMMARY The research was designed to test whether modification of the genetic damage by postirradiation treatment of ir- radiated sperm was possible in males or in inseminated females. Involved in this study was an analysis of the recovery con- cept. Sex-linked lethals and dominant lethals were used as indicators. Comparisons of the percentages of sex—linked or dominant lethals associated with non-stored and stored sperm were made to detect any modifications which occurred. In the first study, Muller-§_fema1es were pre-aged by two methods prior to mating to irradiated males. They were separated into two groups after twenty-four hours of mating. One group was allowed to lay eggs immediately. The other was stored for two weeks at 10°C. After storage, this group was also allowed to lay eggs. In the second study, males were non—stored, stored at room temperature or stored at cold temperature for twenty-four hours. Each set of males was mated to two sets of females and a comparison was made of the difference between first and second-day sperm batches. By starving the females prior and subsequent to mating, an increase in the sex-linked mutation rate was noted. Cold temperature during storage and the aging process of two 35 36 weeks did not, in themselves, contribute to the increase in sex-linked lethals. Evidence from postirradiation storage of males at room temperature and 10°C is presented which does not support a recovery mechanism. It was concluded that the population of mature sperm at the time of irradiation is heterogeneous and compartmentalized with respect to rad- iation sensitivity. On storage at room temperature, mixing of sperm of different radiation sensitivities seemed to occur. Hence, mixing rather than recovery would be respon- sible for the observation that the firstsperm batch of postirradiation-stored males had lower biological damage associated with it than did the first sperm batches of non- stored irradiated males. Cold storage appeared to inhibit mixing, hence the damage associated with cold-stored males' first sperm batch was the same as that of the non-stored males. Cold temperature did not have a statistically significant effect on modifying the sex-linked lethal or dominant lethal rate. However, it is suggested by the parallelism of the cold temperature effect in the sex-linked lethal and dominant lethal studies that postirradiation cold-temperature storage modifies the damage by increasing the sex-linked lethal rate while decreasing the dominant lethal rate. 37 Possible factors contributing to the observed results, such as physiological condition of the female, recovery, mixing of sperm, sperm competition and cold temperature en- hancement of irradiation damage, are discussed. APPENDIX 38 39 Table VII. Experiment II. Detailed results of cold-storing irradiated* sperm in cold prestored females** Non-stored Stored 1 day Stored 14 days 99 1950 2802 3147 56 1689 2596 2990 Sex ratio .5358 .5191 .5128 Normal 1733 2508 2795 Lethal 143 213 239 Sterile 74 81 113 % Lethal 7.62 7.83 7.88 Progeny/9 36 36 32 99/9 20 l9 l6 * 2760 r units of irradiation o ** Prestored on Offerman's medium at 10 C for seven days prior to mating Table VIII. st Experiment IV. 1 Sperm Batch 40 Individual records of sex-linked recessive lethals from NS, SRT, SCT-irradiated males* Non-stored Males (NS) 2nd Sperm Batch 5‘ Normal Lethal %.Lethal Normal Lethal %’Lethal Total % 1 131 6 4.38 65 4 5.78 4.85 2 99 7 6.60 97 3 3.00 4.85 3 124 13 9.49 149 4 2.50 5.86 4 87 12 12.12 27 3 10.00 11.63 5 58 4 6.45 151 7 4.43 5.00 6 160 7 4.19 58 1 1.70 3.54 7 45 3 6.25 15 0 0.00 4.75 8 38 3 7.32 27 0 0.00 4.41 9 89 11 11.00 134 5 3.60 6.69 9 831 66 7.36 723 27 3.60 5.65 Males Stored §£_Room Temperature (SRT) lSt Sperm Batch 2nd Sperm Batch 5‘ Normal Lethal %.Lethal Normal Lethal %1Lethal Total % l 94 3 3.09 118 8 6.35 4.93 2 117 5 4.10 31 2 6.06 4.52 3 55 6 9.84 21 4 16.00 11.63 4 50 2 4.00 90 3 3.23 3.45 5 78 4 4.88 62 2 3.13 4.11 6 57 4 6.55 15 0 0.00 5.26 7 110 11 9.09 69 3 4.17 7.25 8 126 13 9.35 91 5 5.21 7.66 9 148 6 3.90 104 9 7.96 5.62 9 835 54 6.07 601 36 5.65 5.90 41 Con't. Table VIII. Males Stored at Cold Temperature (SCT) St nd Sperm Batch 1 Sperm Batch 2 <3 Normal Letha1 % Lethal Normal Letha1 % Lethal Total % 1 94 7 6.93 43 2 4.44 6.16 2 64 9 12.33 17 3 15.00 12.90 3 105 4 3.67 88 6 6.38 4.92 4 57 8 12.31 106 1 9.35 5.23 5 146 13 8.71 67 3 4.29 6.99 6 105 5 4.55 24 0 0.00 3.73 7 56 2 3.45' 95 6 5.94 5.03 8 94 6 6.00 81 9 10.00 7.89 9 125 6 4.58 24 1 4.00 4.49 10 120 14 10.45 46 2 4.17 8.79 11 69 5 6.75 21 0 0.00 5.26 12 99 14 14.14 122 7 5.42 8.68 12 1134 93 7.59 734 40 5.17 6.65 * 2760 r units of irradiation 42 Table IX. Experiment V. Individual Records of Dominant Lethals from control, NS, SRT and SCT-irradiated males* Non-stored Control 1St Sperm Batch 2nd Sperm Batch 6 Hatch Non-hatch % Hatch Hatch Non-hatch %.Hatch 1 27 4 87.09 91 3 96.8 2 51 5 91.07 94 8 92.2 3 47 2 95.91 57 10 85.7 4 33 6 84.61 84 10 89.4 5 36 1 97.29 81 7 92.04 6 45 3 93.75 60 4 93.75 7 39 8 82.97 57 8 87.69 8 66 5 92.95 12 4 75.00 9 21 0 100.00 46 7 86.72 9 365 34 91.5 582 61 90.5 Non-stored Irradiated lSt Sperm Batch 2nd Sperm Batch 9 Hatch Non-hatch % Hatch Hatch Non-hatch % Hatch 1 19 24 44.19 18 10 64.28 2 24 30 44.44 43 26 62.31 3 38 32 54.28 45 24 65.21 4 36 32 52.94 16 6 72.72 5 20 26 43.47 41 34 54.66 6 30 29 50.84 52 41 55.91 7 25 20 55.55 46 33 58.22 8 34 26 56.66 20 15 57.14 9 35 31 53.03 71 32 68.93 10 25 20 55.55 28 26 51.85 10 286 270 51.4 380 247 60.6 43 Con't. Table IX. Irradiated Stored Room Temperature 1St Sperm Batch 2nd Sperm Batch 5 Hatch Non-hatch % Hatch Hatch Non-hatch % Hatch 1 37 32 53.62 76 71 51.70 2 47 45 51.09 62 55 52.99 3 43 37 53.75 100 59 62.89 4 37 22 62.71 102 70 59.30 5 30 19 61.22 67 49 57.75 6 34 44 43.58 67 46 59.29 7 46 31 59.84 107 55 66.05 8 33 22 60.00 118 81 59.30 9 32 35 47.76 72 44 62.07 10 38 54 41.30 94 70 57.32 10 377 341 52.50 865 600 59.04 Irradiated Stored (10°C) lSt Sperm Batch 2nd Sperm Batch Hatch Non-hatch ‘% Hatch Hatch Non-hatch %1Hatch 1 74 59 55.63 76 29 72.38 2 77 53 59.23 92 72 56.09 3 43 27 61.42 33 20 62.26 4 43 38 53.08 23 13 63.88 5 51 61 45.53 48 27 64.00 6 32 33 49.23 32 21 60.37 7 44 23 65.67 54 19 73.97 8 15 22 40.54 8 9 47.05 9 46 30 60.52 30 21 58.82 9 425 346 55.1 396 231 63.2 * 2760 r units of irradiation Table X. Results of t test on dominant experiment V. 44 lethal data of t P D.F. 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