Differential Effects of Low and High Radiation Dose Rates on Mouse Spermatogenesis.

The adverse effects of radiation are proportional to the total dose and dose rate. We aimed to investigate the effects of radiation dose rate on different organs in mice. The mice were subjected to low dose rate (LDR, ~3.4 mGy/h) and high dose rate (HDR, ~51 Gy/h) radiation. LDR radiation caused severe tissue toxicity, as observed in the histological analysis of testis. It adversely influenced sperm production, including sperm count and motility, and induced greater sperm abnormalities. The expression of markers of early stage spermatogonial stem cells, such as Plzf, c-Kit, and Oct4, decreased significantly after LDR irradiation, compared to that following exposure of HDR radiation, in qPCR analysis. The compositional ratios of all stages of spermatogonia and meiotic cells, except round spermatid, were considerably reduced by LDR in FACS analysis. Therefore, LDR radiation caused more adverse testicular damage than that by HDR radiation, contrary to the response observed in other organs. Therefore, the dose rate of radiation may have differential effects, depending on the organ; it is necessary to evaluate the effect of radiation in terms of radiation dose, dose rate, organ type, and other conditions.

Radiation dosimetry and repair kinetics of DNA damage foci in mouse pachytene spermatocyte and round spermatid stages.

Accurate quantification of DNA double strand breaks (DSB) in testicular germ cells is difficult because of cellular heterogeneity and the presence of endogenous γH2AX. Here, we used confocal microscopy to quantify DNA damage and repair kinetics following γ-irradiation (0.5-4 Gy) in three major mouse male germ cell stages, early and late pachytene spermatocytes and round spermatids (RSs), following a defined post irradiation time course. Dose-response curves showing linear best fit validated γH2AX focus as a rapid biodosimetric tool in these substages in response to whole body in vivo exposure. Stage specific foci yield/dose and repair kinetics demonstrated differential radiosensitivity and repair efficiency: early pachytenes (EP) repaired most rapidly and completely followed by late pachytene (LP) and RSs. Repair kinetics for all three stages followed 'exponential decay' in response to each radiation dose. In pachytenes immediate colocalisation of γH2AX and 53BP1, which participates in non-homologous end-joining repair pathway, was followed by dissociation from the major focal area of γH2AX by 4 h demonstrating ongoing DSB repair. These results confirm the differential radiosensitivity and repair kinetics of DSBs in male germ cells at different stages. Taken together, our results provide a simple and accurate method for assessing DNA damage and repair kinetics during spermatogenesis.

DNA Double Strand Break Response and Limited Repair Capacity in Mouse Elongated Spermatids.

Spermatids are extremely sensitive to genotoxic exposures since during spermiogenesis only error-prone non homologous end joining (NHEJ) repair pathways are available. Hence, genomic damage may accumulate in sperm and be transmitted to the zygote. Indirect, delayed DNA fragmentation and lesions associated with apoptotic-like processes have been observed during spermatid elongation, 27 days after irradiation. The proliferating spermatogonia and early meiotic prophase cells have been suggested to retain a memory of a radiation insult leading later to this delayed fragmentation. Here, we used meiotic spread preparations to localize phosphorylate histone H2 variant (γ-H2AX) foci marking DNA double strand breaks (DSBs) in elongated spermatids. This technique enabled us to determine the background level of DSB foci in elongated spermatids of RAD54/RAD54B double knockout (dko) mice, severe combined immunodeficiency SCID mice, and poly adenosine diphosphate (ADP)-ribose polymerase 1 (PARP1) inhibitor (DPQ)-treated mice to compare them with the appropriate wild type controls. The repair kinetics data and the protein expression patterns observed indicate that the conventional NHEJ repair pathway is not available for elongated spermatids to repair the programmed and the IR-induced DSBs, reflecting the limited repair capacity of these cells. However, although elongated spermatids express the proteins of the alternative NHEJ, PARP1-inhibition had no effect on the repair kinetics after IR, suggesting that DNA damage may be passed onto sperm. Finally, our genetic mutant analysis suggests that an incomplete or defective meiotic recombinational repair of Spo11-induced DSBs may lead to a carry-over of the DSB damage or induce a delayed nuclear fragmentation during the sensitive programmed chromatin remodeling occurring in elongated spermatids.

Effects of testosterone treatment on recovery of rat spermatogenesis after irradiation.

OBJECTIVE: To determine the effects of two different radiation doses on sperm parameters and the role of testosterone treatment on rat spermatogenesis. METHODS: The experimental animal study was conducted at Marmara University, Istanbul, Turkey, from September 2012 to January 2013. Male Sprague Dawley 4-6 months old rats weighing 300-350g were randomely divided into 5 equal groups as control, low dose irradiation, testosterone administration following low dose irradiation, high dose irradiation, and testosterone administration following high dose irradiation. The animals were kept at a constant temperature in a room with 12h light and dark cycles. After the group-wise intervention, sperm concentration, testicular size, and histopathological examination of seminiferous tubules were noted. SPSS 10 was used for statistical analysis. RESULTS: The 40 rats in the study were divided in 5 groups of 8(20%) each. In low dose radiation, adverse effects were only temporarily observed with the return of almost normal testicular function at the end of two months with or without testosterone supplementation. In contrast, in high dose radiation, hormonal treatment effect was controversial. CONCLUSIONS: Testosterone treatment had no significant effect upon recovery after irradiation. In order to prevent the untoward effects of radiation, shielding of the remaining testis in a proper manner is crucial to avoid the harmful effects of the scattered radiation.

[Comparative analysis of ionizing radiation and xenobiotics influence on spermatogenic epithelium and dominant lethal mutations output in laboratory animals].

The study covered state of spermatogenic epithelium and dominant lethal mutations output in mice of BALB/c and CBA lines, subjected to total gamma-irradiation and in Wistar rats after intraperitoneal injection of potassium bichromate (K2Cr2,O7) in small and sublethal doses. The BALB/c line mice under low irradiation dose (0.25 Gy) demonstrated stimulation effect on spermatogenic epithelium, but in the CBA line mice no such effect was seen. Both mice lines under irradiation of 0.25 Gy and 1.0 Gy demonstrated increase in pathologic sperm counts and in percentage ofpreimplantation embryonal death. In rats, injection of potassium bichromate in doses of 0.028 mg/kg and 2.8 mg/kg increased number of micronuclear spermatids, larger pathologic sperm counts and percentage of postimplantation deaths. Thus, lower general embryonal deaths under radiation exposure is due to preimplantation embryonal deaths, under exposure to 6-valent chromium--is due to postimplantation losses.

Transcriptome profiling of mice testes following low dose irradiation.

BACKGROUND: Radiotherapy is used routinely to treat testicular cancer. Testicular cells vary in radio-sensitivity and the aim of this study was to investigate cellular and molecular changes caused by low dose irradiation of mice testis and to identify transcripts from different cell types in the adult testis. METHODS: Transcriptome profiling was performed on total RNA from testes sampled at various time points (n = 17) after 1 Gy of irradiation. Transcripts displaying large overall expression changes during the time series, but small expression changes between neighbouring time points were selected for further analysis. These transcripts were separated into clusters and their cellular origin was determined. Immunohistochemistry and in silico quantification was further used to study cellular changes post-irradiation (pi). RESULTS: We identified a subset of transcripts (n = 988) where changes in expression pi can be explained by changes in cellularity. We separated the transcripts into five unique clusters that we associated with spermatogonia, spermatocytes, early spermatids, late spermatids and somatic cells, respectively. Transcripts in the somatic cell cluster showed large changes in expression pi, mainly caused by changes in cellularity. Further investigations revealed that the low dose irradiation seemed to cause Leydig cell hyperplasia, which contributed to the detected expression changes in the somatic cell cluster. CONCLUSIONS: The five clusters represent gene expression in distinct cell types of the adult testis. We observed large expression changes in the somatic cell profile, which mainly could be attributed to changes in cellularity, but hyperplasia of Leydig cells may also play a role. We speculate that the possible hyperplasia may be caused by lower testosterone production and inadequate inhibin signalling due to missing germ cells.

Parp1-XRCC1 and the repair of DNA double strand breaks in mouse round spermatids.

The repair of DNA double strand breaks (DSBs) in male germ cells is slower and differently regulated compared to that in somatic cells. Round spermatids show DSB repair and are radioresistant to apoptosis induction. Mutation induction studies using ionizing irradiation, indicated a high frequency of chromosome aberrations (CA) in the next generation. Since they are in a G1 comparable stage of the cell cycle, haploid spermatids are expected to repair DSBs by the non-homologous end-joining pathway (NHEJ). However, immunohistochemical evidence indicates that not all components of the classical NHEJ pathway are available since the presence of DNA-PKcs cannot be shown. Here, we demonstrate that round spermatids, as well as most other types of male germ cells express both Parp1 and XRCC1. Therefore, we have determined whether the alternative Parp1/XRCC1 dependent NHEJ pathway is active in these nuclei and also have tested for classical NHEJ activity by a genetic method. To evaluate DSB repair in SCID mice, deficient for DNA-PKcs, and to study the involvement of the Parp1/XRCC1 dependent NHEJ pathway in round spermatids, the loss of gamma-H2AX foci after irradiation has been determined in nucleus spreads of round spermatids of SCID mice and in nucleus spreads and histological sections of Parp1-inhibited mice and their respective controls. Results show that around half of the breaks in randomly selected round spermatids are repaired between 1 and 8h after irradiation. The repair of 16% of the induced DSBs requires DNA-PKcs and 21% Parp1. Foci numbers in the Parp1-inhibited testes tend to be higher in spermatids of all epithelial stages reaching significance in stages I-III which indicates an active Parp1/XRCC1 pathway in round spermatids and a decreased repair capacity in later round spermatid stages. In Parp1-inhibited SCID mice only 14.5% of the breaks were repaired 8h after irradiation indicating additivity of the two NHEJ pathways in round spermatids.

[The alterations in profiles of RAPD- and of ISSR-markers in progeny of the single gamma-irradiated mice of BALB/c strain].

Molecular-genetic effects in the offspring of BALB/c male mice exposed to single radiation doses of 1, 2 and 3 Gy were studied. Induced genetic variability was studied using such methods as assessment of variation RAPD- and ISSR-profiles. Comparative analysis of genetic radiosensitivity of stem spermatogonia and of spermatids is presented in the work. The frequency of changes in the patterns of the offsprings of irradiated mice was significantly different from the analogous parameters in the offsprings of the control group already at a dose of 1 Gy. Comparative analysis of genetic radiosensitivity at different stages of spermatogenesis revealed the similar sensitivity of spermatogonia and of spermatids at 1 and 3 Gy and a higer sensitivity of spematogonia at 2 Gy.

[The influence of chronic and of acute irradiation on the polymorphism of RAPD-markers in offspring for irradiated mice].

On mice lines BALB/c and CBA/lac was performed the study of molecular-genetics effects in mice progeny after the chronic (dose rate -0.0017 Gy/day, total dose -0.36 Gy) and acute (dose range 1-3 Gy) exposure of y-radiation on the parents. For variability analysis was used technique of amplification DNA with series of random primers (RAPD-assay). Random primers were used as single primer and in mixture of ones. In this work were held the comparative analysis of the genetic radiosensitivity for stem spermatogonia and spermatides. After the acute exposure the dose dependence for levels of polymorphism of RAPD-markers were obtained. After the chronic irradiation, significant differences from control group were obtained only by use primers mixture M1. Comparative analysis of the genetic radiosensitivity of different stages of mice spermatogenesis are display is similar sensitivity of stem spermatogonia and spermatides after doses of irradiation 1 Gy and 3 Gy. Indicated that after irradiation by dose 2 Gy, spermatogonia are more sensitivity than spermatides.

Assessment of testicular function after acute and chronic irradiation: further evidence for an influence of late spermatids on Sertoli cell function in the adult rat.

To study cell to cell communications within the testis of adult Sprague-Dawley rats, we used acute whole body neutron plus gamma-irradiation (0.99 Gray of neutron and 0.24 Gray of gamma-rays, 3 min; Exp A) over 7-121 days postirradiation and chronic whole body gamma-irradiation (7 cGy/day 60Co gamma-rays; Exp B) over 14-84 days of irradiation and 7-86 days postirradiation. Neither irradiation protocol had an effect on the body weight of the animals. Neutron plus gamma-rays induced dramatic damages to spermatogonia, preleptotene spermatocytes, spermatozoa, and, to a lesser extent, pachytene spermatocytes. In contrast, gamma-rays induced a selective destruction of spermatogonia. Subsequently, in both experiments a maturation-depletion process led to a marked decrease in all germ cell types. A complete or near complete recovery of the different germ cell types and spermatozoa took place during the two postirradiation periods. Under both irradiation protocols Sertoli cells number was unchanged. Androgen-binding protein and FSH levels were normal in spite of the disappearance of most germ cells from spermatogonia to early spermatids. However, the decline of androgen-binding protein as well as the rise of FSH and their subsequent recovery were highly correlated to the number of late spermatids and spermatozoa. Moreover, it appeared that spermatocytes may also interfere with the production of inhibin (Exp B). With neither irradiation was Leydig cell function altered, except in Exp B in which elevated LH levels were temporarily observed. Correlation analysis suggested a relationship between preleptotene spermatocytes and Leydig cell function. In conclusion, this study establishes that chronic gamma-irradiation is particularly useful in the study of intratesticular paracrine regulation in vivo and provides further support to the concept that late spermatids play a major role in controlling some aspects of Sertoli cell function in the adult rat.

In vivo effects of iron-55 and iron-59 on mouse testes: biophysical dosimetry of Auger electrons.

When the Auger-electron emitter, 55Fe, and the beta-emitter, 59Fe, are similarly distributed in the testes of mice, the conventionally calculated average radiation dose to the organ from 55Fe is about 2.6 times more effective in reducing the sperm-head count than the dose from 59Fe. This finding emphasizes the ability of low-energy Auger electrons to damage radiosensitive targets of cells through localized irradiation. The observed efficacy is understandable in terms of dosimetric models based on intracellular distribution studies of the radionuclides.

Stage-dependent variation in the radiosensitivity of DNA in developing male germ cells.

The induction and rejoining of gamma-ray-induced DNA single-strand breaks (SSBs) were measured in the spermatogenic cells of mice using the alkaline elution technique. The animals were injected with [3H]thymidine and sacrificed on subsequent days to examine selectively cohorts of radiolabeled cells in the successive stages of maturation. A significantly increased frequency of SSB was observed in the unirradiated early spermatocytes and late spermatids, associated with genetic recombination and chromatin compaction, respectively. The frequency of SSBs induced by irradiation of animals in vivo remained constant from the early spermatocyte through mid-spermatid stages and decreased significantly only after the cells matured to the late spermatid stage. The frequency of SSBs after in vitro irradiation of testicular cell suspensions also decreased as round spermatids matured to late spermatids. Such decreases for both modes of irradiation may result from maturation-dependent alterations in chromatin in late spermatids, such as condensation and replacement of histones with protamines, rather than from changes in oxygen tension. Rejoining of SSBs in vivo was efficient in the spermatocytes and early spermatids but declined in late spermatids. Possible reasons for the discrepancy between the greater number of unrepaired lesions and lower susceptibility to mutation induction in late spermatids than in round spermatids are discussed.

Effects of single and split doses of cobalt-60 gamma rays and 14 MeV neutrons on mouse stem cell spermatogonia.

The long-term effects of ionizing radiation on male gonads may be the result of damage to spermatogonial stem cells. Doses of 10 cGy to 15 Gy (60)Co gamma rays or 10 cGy to 7 Gy 14 MeV neutrons were given to NMRI mice as single or split doses separated by a 24-h interval. The ratios of haploid spermatids/2c cells and the coefficients of variation of DNA histogram peaks as measures of both the cytocidal and the clastogenic actions of radiation were analyzed by DNA flow cytometry after DAPI staining. The coefficient of variation is not only a statistical examination of the data but is also used here as a measure of residual damage to DNA (i.e. a biological dosimeter). Testicular histology was examined in parallel. At 70 days after irradiation, the relative biological effectiveness for neutrons at 50% survival of spermatogonial stem cells was 3.6 for single doses and 2.8 for split doses. The average coefficient of variation of unirradiated controls of elongated spermatids was doubled when stem cells were irradiated with single doses of approximately 14 Gy (60)Co gamma rays or 3 Gy neutrons and observed 70 days later. Split doses of (60)Co gamma rays were more effective than single doses, doubling DNA dispersion at 7 Gy. No fractionation effect was found with neutrons with coefficients of variation.

Course of c-myc mRNA expression in the regenerating mouse testis determined by competitive reverse transcriptase polymerase chain reaction.

The c-myc proto-oncogene is a reliable marker of the "G0-early G1" transition, and its down-regulation is believed to be necessary to obtain cellular differentiation. In murine spermatogenesis, the level of c-myc transcripts does not correlate with the rate of cellular division. Proliferation of supposed staminal spermatogonia to reproduce themselves is induced with a local 5 Gy X-ray dose in 90-day-old C57Bl/6 mice. c-myc quantification by a newly developed competitive reverse transcriptase polymerase chain reaction (RT-PCR) was carried out to follow the expression course of this proto-oncogene. Damage and restoration of spermatogenesis were analyzed at days 3, 6, 9, 10, 13, 30, and 60 after injury by relative testes/body weight determination and histological examination. Proliferative status was determined by histone H3 Northern blot analysis. c-myc mRNA level was 10 times higher after 3 days in the irradiated animals compared to the controls. An increasing number of copies were noted up to 10 days, but promptly decreased to the base level found for irradiated mice from 13 to 60 days. Interestingly, the expression of histone H3 detected S phase only in testes at 60 days from damage.

Radiation-induced diploid spermatids in mice.

Diploid elongated spermatids of mice were enriched by flow cytometry and cell sorting using a new type of sorter (Partec). The sorted abnormal spermatids were identified morphologically and by nuclear area integration. The radiation-induced increase in the frequency of diploid elongated spermatids was monitored with time following acute X-ray exposure of mice. Dose-response curves for acute 60Co-gamma and 14 MeV neutron irradiations yielded an RBE value of 4.3 for the doubling of the control level.

Dose-response of a radiation induction of a germline mutation at a hypervariable mouse minisatellite locus.

Dose-response of an induction of a germline mutation was studied at a hypervariable mouse minisatellite locus, Ms6hm, which consists of tandem repeats of a sequence motif GGGCA. Male C3H/HeN mice were exposed to various doses of 60Co gamma-ray and mated with unirradiated C57BL/6N female mice. Matings were done at various time after irradiation to assess the effects of radiation on spermatozoa, spermatids and spermatogonia. DNA samples of F1 offsprings were analysed by Southern blotting for the repeat length mutation at the Ms6hm locus. The mutation frequency per gamete of the paternal allele was 9.1% for the unirradiated control group. The spermatids stage was most sensitive to radiation and a statistically significant dose-response was observed. The mutation frequency of the paternal allele in F1 mice increased to 22% for 1 Gy, 28% for 2 Gy, and 28% for 3 Gy. The spermatogonia stage was less sensitive to radiation, and the mutation frequencies of the paternal allele were 14% for 2 Gy, and 16% for 3 Gy. The spermatozoa stage germ cells were also less sensitive and the frequency of mutation of the paternal allele increased to 14% for 3 Gy. However, these increases were statistically not significant. Possible mechanisms of radiation induction of germline mutation at the hypervariable minisatellite locus will be discussed.

Radiation induction of germline mutation at a hypervariable mouse minisatellite locus.

Paternal 60Co gamma-irradiation was tested for the induction of germline mutation at the mouse hypervariable minisatellite locus, Ms6hm. Male C3H/HeN mice were exposed to 3 Gy 60Co gamma-ray and mated with C57BL/6N females. Matings were made at 1-7, 15-21 and 71-77 days post-treatment to test spermatozoa, spermatids and spermatogonia stages. Reciprocal crosses were also made with irradiated C57BL/6N males. Southern analysis was carried out on DNA from parents and F1 mice. The paternal mutation frequencies per gamete of the Ms6hm locus were 8.3, 13, 28 and 15% for the C3H/HeN control, exposed spermatozoa, spermatids and spermatogonia stages, respectively. The paternal mutation frequencies per gamete were 7.7% for the C57BL/6N control and 13% for the C57BL/6N exposed spermatozoa stage. The increase in the paternal germline mutation frequency was statistically significant for C3H/HeN spermatids irradiation (p < 0.005). The induced mutation frequencies were of the order of 10(-1), and was too high to be accounted for by the direct action of radiation on the locus. These results suggest the presence of a previously unexpected mechanism of radiation induction of germline mutation. In addition, we demonstrate that the hypervariable minisatellite locus can serve as a sensitive monitor for genetic damages to germline cells.

Influence of oxygen on radiation-induced DNA damage in testicular cells of C3H mice.

Radiation-induced DNA single-strand break (ssb) induction and rejoining were measured in murine testicular cells using the alkaline comet assay. Individual cells in different stages of differentiation were identified on the basis of DNA content. As expected, induction of DNA ssb in testis cells irradiated on ice was independent of ploidy, and the extent of damage was similar to that produced in cells from other normal tissues. However, in vivo irradiation of air-breathing mice produced more ssb in haploid than tetraploid germ cells, although their rates of rejoining were similar and comparable to repair rates of cells from other normal tissues. In addition, irradiation of testis in situ produced only half as much damage as irradiation in vitro, and this could be explained only in part by the rapid ssb rejoining occurring during irradiation and cell isolation. A lower cellular oxygenation was postulated to account for the apparent resistance of testis cells to induction of breaks and the difference in induction in relation to DNA content. This was confirmed when carbogen inhalation and treatment with nicotinamide not only increased the overall degree of ssb induction in all these cells, but also reduced differences between cells of different ploidies. Results using the hypoxic cell cytotoxin RSU 1069 confirmed that the extent of hypoxia was not as severe in the testis as in the SCCVII murine tumour. It can be concluded from these data that the oxygenation of all testis cells is low enough to confer radioresistance, and that haploid testis cells are less hypoxic than tetraploid spermatocytes.

Influence of teniposide (VM-26) on radiation-induced damage to mouse spermatogenesis: a flow cytometric evaluation.

The effect of teniposide (VM-26) 0.05 mg/kg body weight treatment on spermatogenesis of mice exposed to 0, 0.5, 1, 2, and 3 Gy gamma-radiation was evaluated flow cytometrically. Whole body irradiation with 1 to 3 Gy resulted in a significant decline in testis weight from Day 14 to 35 post-irradiation depending on the exposure dose. Treatment of mice with teniposide before irradiation advanced the decline in testicular weight by several days, especially at 3 Gy, where a significant decline in testicular weight was observed at Day 7 post-irradiation when compared with the double distilled water (DDW)+irradiation group. The relative percentage of the 2C population declined significantly in the VM-26+irradiation group in comparison with the DDW+irradiation group at various post-irradiation time periods depending on the exposure dose. A significant depletion in the relative percentage of S-phase cells was observed as early as Day 1 post-irradiation in the VM-26+irradiation group when compared with the DDW+irradiation group after exposure to 1 to 3 Gy. This decline continued up to Day 21 post-irradiation after exposure to 2 Gy. The relative percentage of primary spermatocytes showed a consistent decline after exposure to various doses of gamma-radiation in the VM-26+irradiation group when compared with the DDW+irradiation group at different time periods, with a few exceptions, especially at higher doses. The pattern of decline in the relative percentage of round spermatids was similar to that of primary spermatocytes, where a significant decline was observed at various post-irradiation time periods in the VM-26+irradiation group in comparison with the DDW+irradiation group. These changes in the relative germ cell percentages are manifested as alterations in the ratios of various germ cell populations. The 4C:2C ratio declined consistently from Day 1 to Day 70 post-irradiation in the VM-26+irradiation group at all exposure doses. Similarly, the 4C:S-phase ratio in the VM-26+irradiation group also showed a significant decline at different post-irradiation time periods when compared with the DDW+irradiation group depending on the exposure dose. The reduction observed in the relative percentages of various cell populations was higher in the combination group when compared with the DDW+irradiation controls, indicating potentiation of damage to male germ cells by teniposide treatment before irradiation.

The induction of sex-chromosomal nondisjunction and diploid spermatids following x-irradiation of pre-spermatid stages in the northern vole Microtus oeconomus.

Chromosome nondisjunction seems to be one of the most important mutagenic effects occurring in man and makes an enormous contribution to human foetal wastage. As yet, little or no information is available on which environmental factors are important in inducing nondisjunction and accordingly we have investigated the effect of X-irradiation on inducing nondisjunction in male germ cells of an experimental mammal, the Northern vole-Microtus oeconomus. Using a staining technique based upon the presence of heterochromatin we have scored the number of sex chromosomes in early spermatids in both irradiated and unirradiated animals. A significant increase in nondisjunction, following treatment, was found with all doses between 25 and 200 R. However, variations in nondisjunction induction at various time intervals following irradiation suggest variations in cell stage sensitivity. More surprising was the large induction of diploid gametes which also demonstrated a significant induction with all irradiation doses. From the distribution of sex chromosomes we conclude that both nondisjunction and diploid gamete induction occur at both meiotic divisions. At present it is not possible to conclude whether the radiation response is linear and to define the cell-stage sensitivity with precision. The reasons for this appear to be variations in sensitivity between animals and also that there is a clear overlap between the duration of the early spermatid stage analyzed (4 days) and the interval between sampling times.