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.

Aging does not affect spermatogenic recovery after experimentally induced injury in mice.

Testes in aging mammals undergo a variety of age-related changes, such as reduction of size, lower sperm output, an increase in abnormal forms of sperm, and endocrine malfunctions. It has been suggested that the spermatogenic defects are due to loss and dysfunction of spermatogonial stem cells as well as deterioration of the tubule microenvironment. In the present study, we explore the depletion and recovery of spermatogenesis in young (3 month) and old (12 month) mice exposed to cooling, X-irradiation (5 Gy) or cytotoxic treatment using Busulfan (40 mg/kg). We aim to determine a potential age-related change of vulnerability to gonadotoxic treatments by describing the intensity of spermatogenic depletion and the degree of spermatogenic recolonization with qualitative and quantitative parameters on organ weights and histological parameters at two time points (2 weeks, depletion; 6 weeks, recovery). Our data reveal specific acute effects of cooling on multinucleation of germ cells but no other severe injury. Irradiation and Busulfan-treatment exerted the expected depletional wave of germ cells leading to severe testicular injury and spermatogenic failure. The recovery of spermatogenesis occurred in both treatment groups and both age groups to a similar extent. We therefore noted no prominent age-related differences in spermatogenic depletion and recovery in any treatment group. We conclude that in both age groups, the remaining spermatogonial stem cells are capable to induce spermatogenic recovery and the aging tubule microenvironment at 1 year has not become more vulnerable to irradiation, Busulfan-treatment or testicular cooling.

[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.

Radio-protective effect of vitamin E on spermatogenesis in mice exposed to gamma-irradiation: a flow cytometric study.

AIM: To investigate the effect of vitamin E on the radioprotection of spermatogenesis and chromatin condensation of spermatozoa during passage through the epididymis in mice exposed to irradiation. METHODS: Adult outbred male ICR mice were orally administered natural vitamin E (VE, D-alpha-tocopheryl acetate) at 400 IU/kg for 7 days before exposure to 1 Gy of gamma-irradiation. The animals were sacrificed at day 1, 7, 14, 21, 28, 35 and 70 post-irradiation (IR) and the percentage of testicular germ cells and epididymal sperm chromatin condensation was analyzed using flow cytometry. RESULTS: Serum D-alpha-tocopheryl acetate levels were 47.4+/-3.2 microg/dL in the treated group, yet it could not be detected in the control group. The testicular weight of irradiated mice pretreated with VE+IR was significantly (P<0.05) higher than that of those without VE treatment (IR) at day 14 and 21 post-irradiation. The percentage of primary spermatocytes (4C) in the VE+IR group was comparable to the controls but significantly (P<0.05) higher than those in the IR group from day 7 to 35 post-irradiation. The percentage of round spermatids (1C) in the VE+IR group was also significantly (P<0.05) higher than those in the IR group at day 28 post-irradiation. The primary spermatocytes : spermatogonia ratio in the IR group was significantly (P<0.05) declined at day 7 to 35 post-irradiation when compared to the VE+IR and control groups. The round spermatid : spermatogonia ratio in the VE+IR group was significantly (P<0.05) higher than that of the IR group at day 14 and 28 post-irradiation. The chromatin condensation of epididymal spermatozoa measured by propidium iodide uptake was not affected by 1 Gy of gamma-irradiation. CONCLUSION: The administration of VE prior to irradiation protects spermatogenic cells from radiation.

Transgenerational genomic instability as revealed by a somatic mutation assay using the medaka fish.

We previously established a somatic mutation assay of the medaka wl (white leucophores) locus based on visual inspection, and showed that somatic mutations at paternally derived alleles frequently arise during the development of F1 embryos fertilized by sperm/late spermatids that had been exposed to gamma-rays. To further study such delayed mutations, we determined the frequency of mutant embryos obtained from three different crosses between irradiated males and non-irradiated females. When sperm and late spermatids were irradiated, the mutant frequency within non-irradiated maternally derived alleles was approximately 3 times higher than in the control group. In the F2 generation, however, no increase in mutant frequency was observed. Similarly, there was no significant increase in the F1 mutant frequency when stem spermatogonia were irradiated. These data suggest that irradiation of sperm and late spermatids can induce indirect mutations in F1 somatic cells, supporting the idea that genomic instability arises during F1 embryonic development. Moreover, such instability apparently arises most frequently when eggs are fertilized just after the sperm are irradiated.

Parental exposure to low-dose X-rays in Drosophila melanogaster induces early emergence in offspring, which can be modulated by transplantation of polar cytoplasm.

In recent years there has been growing concern over the biological effects of low-dose X-rays, but few studies have addressed this issue. Our laboratory had observed flies (Drosophila melanogaster) irradiated with low-dose X-rays tend to emerge earlier than normal flies. This observation led us to quantitatively examine the effects of low-dose X-irradiation on development in the fly. Following exposure of prepupal (day 5) flies to 0.5 Gy X-rays, the time to emergence was slightly shorter than in the sham controls. This tendency was increased when the X-ray exposure came during the pupal stage (day 7). In these flies, the time to eclosion decreased significantly, by an average of 30 h sooner than sham controls. A further experiment examined whether such radiation effects could be observed in the unexposed F1 generation of exposed individuals. Greater radiation effects on early F1 emergence were seen when the time between exposure and mating was 3 days, indicating an effect on early spermatid development. Early F1 emergence was also observed after exposure of female flies to X-rays during late previtellogeny. Furthermore, rapid emergence could be induced in the F1 embryos of unexposed parents by transferring the polar cytoplasm (precursor cells of the germ cell line) from F1 embryos of exposed flies. These results show that radiation-induced effects can be transmitted to the next generation through the germ cell line.

[Morphofunctional state of the rat male reproductive system after chronic low intensity irradiation at a dose of 1.0 Gy]. / Morfofunktsional'noe sostoianie reproduktivnoi sistemy krys-samtsov posle khronicheskogo nizkointensivnogo oblucheniia v doze 1.0 Gr.

It was studied the morphofunctional state of rat male reproductive system after chronically exposure to low intensive gamma-irradiation in early ontogenesis. After radiation with a total dose of 1.0 Gy it was revealed the decrease of the testes and epididymis weight, dysbalance in the ratio of different spermatogenic cells, changes in nucleic acid content and discoordination of bioenergetic metabolism in testes. Disturbance in rat male reproductive system retained in remote period (180 days after irradiation).

Fas is involved in the p53-dependent apoptotic response to ionizing radiation in mouse testis.

Apoptosis induced in male germ cells following ionizing radiation is dependent on functional p53 (Trp53) being present. We sought to determine whether Fas (Tnfrsf6/CD95/APO-1), an apoptotic factor, is involved in this p53-dependent germ cell death. In p53 knock-out mice exposed to 5 Gy of x-radiation, germ cells were protected from cell death, as assessed by counting apoptotic seminiferous tubules 12 h following radiation. Similarly, spermatid head counts in p53 knock-out mice remained near normal 29 days after exposure to 0.5 Gy of radiation, whereas wild-type animals had a more than twofold reduction in spermatid head counts. Fas mRNA expression remained at pretreatment levels in p53 knock-out mice; however, Fas increased in a time-dependent manner in wild-type mice following exposure to 5 Gy of radiation, indicating that radiation-induced Fas expression is p53-dependent. The functional significance of Fas involvement was demonstrated when lpr(cg) mice, having a nonfunctional Fas receptor, were exposed to 5 Gy of radiation; the number of apoptotic seminiferous tubules 12 h following radiation was significantly reduced compared to that of wild-type mice. Additionally, lpr(cg) mice exposed to 0.5 Gy of radiation had increased spermatid head counts 29 days following radiation compared to wild-type mice. Interestingly, gld mice with a non-functional Fas ligand (Tnfsf6/FasL/CD95L) were as sensitive to radiation as wild-type animals, and levels of FasL mRNA were not affected by radiation treatment. These results indicate that apoptosis and up-regulation of Fas following radiation are both p53-dependent events. Although Fas is necessary, in part, for radiation-induced p53-dependent apoptosis, FasL is not.

Micronuclei induced by low dose rate irradiation in early spermatids of p53 null and wild mice.

To obtain evidence of the dose-rate effect on induction of micronuclei in early spermatids, we observed frequencies in wild-type p53(+/+), heterozygous p53(+/-) and null p53(-/-) mice 14 days after gamma rays irradiation at a high (1,020 mGy/min) or a low (1.2 mGy/min) dose-rate. A dose- and dose-rate-related increase in micronuclei was seen in early spermatids with no difference between the different p53 status. These data were found to be best fitted by a linear-quadratic dose-response model at a high dose-rate, and by a linear dose-response model at a low dose-rate. The yields at 1.2 mGy/min were significantly lower than those at 1,020 mGy/min in the same manner, independent of p53 status. Testis weight declined significantly after 3 Gy irradiation, but did not depend on dose-rates. In our other studies, we observed the complete elimination both of malformation in fetuses and CD3- 4+ mutant T-lymphocytes in p53(+/+) mice, but not in p53(-/-) mice after irradiation. This indicates that concerted DNA repair and p53-dependent apoptosis are likely to completely eliminate mutagenic damage from the irradiated tissues at low doses or dose-rates in teratogenesis and lymphocytes. In the germ cell, however, irradiation at 1.2 mGy/min was mutagenic, independent of p53 status.

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.

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.

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.

Heritable malformations in the progeny of the male medaka (Oryzias latipes) irradiated with X-rays.

Heritable malformations were examined in the progeny of X-irradiated male medaka (Oryzias latipes) by three-generation crosses. Two X-irradiated male fish were pair-mated with non-irradiated females to produce F1 founders, and each F1 fish was pair-mated with a non-irradiated fish to produce F2 progeny. For detection of recessive mutations, pair-matings between F2 siblings were performed for each F1 family. Morphogenesis of the embryos of each generation was observed using a stereomicroscope, throughout the entire period of embryonic development. In the F1 embryos, the frequencies of dominant lethals and malformations were increased by the X-irradiation. Two out of 30 F1 pairs produced a number of malformed and lethal F2 embryos, indicating inheritance of high rates of the dominant lethals in the two F1 families. Moreover, F2 sib-pairs offspring of which exibited high rates of dominant lethals were found in 10 out of 28 F1 families. Recessive lethal mutations, which were associated with a particular phenotype, were found in 2 out of the 28 F1 families. These results indicate that the heritable malformations induced by X-irradiation can be studied in the medaka.