The induction of chromosomal damage and cell killing in mouse spermatogonial stem cells following combined treatments with hydroxyurea, 3-aminobenzamide and X-rays

To analyze in more detail the relation between the sensitivity of spermatogonial stem cells to killing and the induction of genetic damage, mature male mice received combined treatments with hydroxyurea (HU), 3-aminobenzamide (3-AB) and X-rays. Stem cell killing was determined using the repopulation index method and translocations were studied via spermatocyte analysis. HU was administered at 16 or at 48 h before further treatment in order to create stem cell populations with different sensitivities in whic the translocation induction and stem cell killing could be studied and compared. The sensitivities for cell death and genetic damage appeared to be strongly correlated: at 16 h after HU significantly higher values were found than at 48 h or in controls without HU pretreatment.By using 3-AB in the treatment schedules we were able to investigate whether the sensitization of stem cells towards cell death and genetic damage is the outcome of a radiation- or drug-induced G₁ delay. The effect of 3-AB was most pronounced at 16 h after HU. This confirms that at this interval a large fraction of stem cells is in G₁. Our data therefore indicate that all treatments that induce an enrichment of G₁ cells also result in a sensitization of stem cells to cell killing or the induction of mutagenic damage.     

Radioprotective effect of misoprostol on mouse spermatogonial stem cells

The radioprotective effects of misoprostol, a synthetic stable analogue of prostaglandin E1, on spermatogonial stem cells of C3H/HeH x 101/F1 hybrid mice (3H1) were analysed by establishing dose--response relationships for stem cell killing by X-rays in mice that were pretreated with misoprostol. Spermatogonial stem cell killing was studied through determination of the percentage of tubular cross-sections showing repopulation at 10 days after irradiation. In control mice, the D0 values ranged between 1.7 and 3.6 Gy, dependent on the stage of the cycle of the seminiferous epithelium the cells were in. As found previously, proliferating spermatogonial stem cells were much more radioresistant than quiescent stem cells. In the misoprostol-pretreated animals the spermatogonial stem cells were more radioresistant, the D0 values ranging from 3.6 to 5.0 Gy. Both proliferating and quiescent spermatogonial stem cells were protected by misoprostol. As the dose--response curves in control and misoprostol-pretreated mice showed about the same extrapolation number to the y-axis it was concluded that the misoprostol pretreatment did not alter the kinetics of the repopulation process.     

Enhanced recovery of radiation-induced translocations from spermatogonial stem cells of p53 null mice

X-ray-induced (4Gy) chromosomal translocations were studied in mouse spermatogonial stem cells with different p53 status by meiotic analysis at the spermatocyte stage, many cell generations after the moment irradiation. The results show enhanced recovery of translocations from p53 -/- mice relative to +/- and +/+ littermates. The enhanced recovery is probably due to an altered cell cycle distribution of the stem cells in the -/- mice leading to less radioresistant G(0)-G(1) transition cells, rather than differences in apoptotic response. Experiments with the poly(ADP-ribose)polymerase inhibitor 3-aminobenzamide (3-AB) indicate that, in contrast to the situation in +/+ mice, no sensitization in the p53-deficient mice occurred for both testis weight loss and the recovery of induced translocations. This result also points to the presence of less radioresistant stem cells in the testis of p53 null mice.     

X-ray induced translocations in premeiotic germ cells of monkeys

The induction of reciprocal translocations by various X-ray exposures was studied in spermatogonial stem cells of rhesus monkeys (Macaca mulatta) and stump-tailed macaques (Macaca arctoides) by means of spermatocyte analysis many cell generations after irradiation. The yields of translocations recovered from irradiated stump-tailed macaques were lower than those observed in rhesus monkeys and represent in fact the lowest induction rates per Gy ever recorded for experimental mammals. In the rhesus monkey a humped dose-effect relationship was found with (a) a homogeneous response with (pseudo-)linear kinetics below 1 Gy, (b) much more variability at higher doses, and (c) no induction at all at doses of 4 Gy and above. It is suggested that the post-irradiation proliferation differentiation pattern of surviving rhesus monkey spermatogonial stem cells i mainly responsible for these characteristics of the dose-response curve.     

Radiation-induced translocations in spermatogonial stem cells of Macaca fascicularis and Macaca mulatta

3 adult monkeys, one Macaca fascicularis and two Macaca mulatta, were whole-body irradiated with 1 Gy gamma-rays (60 Co). Reciprocal translocations induced in spermatogonial stem cells were scored as translocation multivalents in primary spermatocytes from 7.5 to 27.5 months after exposure. The translocation yields ranged from 4.1% at the earliest to 1.8% at the latest sampling interval. No significant differences were observed in the responses of the individual animals. A decline in the translocation frequencies with time after treatment was found in all 3 animals. The present data are different from those reported for testicular X-irradiation of the rhesus monkey Macaca mulatta (van Buul, 1980; Lyon et al., 1976) in that the translocation yields are higher. They are consistent with the results reported for testicular gamma-irradiation of the crab-eating monkey Macaca fascicularis (Matsuda et al., 1984, 1985). In view of the present results it appears unlikely that a species difference exists within the genus Macaca in the sensitivity of spermatogonial stem cells to the induction of translocations by ionizing radiation.     

γ-Ray-induced reciprocal translocations in spermatogonia of the crab-eating monkey (Macaca fascicularis)

The yield of translocations induced by γ-rays in the crab-eating monkey (Macaca fascicularis) spermatogonia were studied by cytological analysis in spermatocytes derived from them. The frequencies of translocations were 0.09% at 0 Gy, 1.9% at 1 Gy, 2.5% at 2 Gy and 1.3% at 3 Gy, showing a humped dose-response curve with a peak yield around 2 Gy. No remarkable inter-seasonal or inter-animal variations in the induction of translocation were observed. The frequencies in the crab-eating monkey were significantly higher than those in the same Macaca genus, the rhesus monkey (Macaca mulatta) (van Buul, 1976, 1980). This inter-species difference in radiosensitivity might be affected by the condition of spermatogonial stem cells at the time of exposure to radiation, depending on the seasonal change in spermatogenetic activity.     

Enhanced radiosensitivity for the induction of translocations in mouse stem cell spermatogonia following treatment with cyclophosphamide or adriamycin

The effects of pretreatment of mouse spermatogonial stem cells with cyclophosphamide (100 and 200 mg/kg) and adriamycin (2.5, 5 and 7 mg/kg) on the induction of chromosomal translocations by high doses (800 or 900 rad) of X-rays applied 24 h later, were studied by spermatocyte analysis. The results indicate that both compounds were able to alter the chromosomal radiosensitivity of surviving stem cells. It is concluded that depletion of differentiating and differentiated spermatogonia is sufficient for triggering stem cells into a more radiosensitive phase.     

The relation between induced reciprocal translocations and cell killing of mouse spermatogonial stem cells after combined treatments with hydroxyurea and X-rays

The induction of reciprocal translocations in mouse spermatogonial stem cells, visualized in dividing primary spermatocytes, was studied after combined treatments with hydroxyurea (250 and 500 mg/kg) and X-rays (6, 8 and 9 Gy). The time intervals between the 2 treatments were 16 h (leading to extremely high cell killing) and 48 h (giving rise to less killing than irradiation alone). Comparison of the observed frequencies of translocations with reported data on stem cell killing (de Ruiter-Bootsma and Davids, 1981 show that the ratio between the probabilities that a radiation-induced basic lesion kills a cell or produces a translocation, theoretically calculated by Leenhouts and Chadwick (1981) to be about 10, can indeed be confirmed experimentally.     

The response of murine stem spermatogonia to radiation combined with 3-aminobenzamide

The influence of 3-aminobenzamide (3-AB) on the radiation response of the stem spermatogonia of the CBA mouse has been investigated. Doses of 3-AB from 66 to 450 mg/kg, administered 1 h before irradiation, significantly enhanced stem-cell killing. Enhancement was observed when 3-AB (450 mg/kg) was given up to 5 h before, but not if administered after, irradiation. When radiation was delivered at a lower dose rate (5 cGy/min compared to 180 cGy/min) significant dose sparing was achieved for radiation alone. Pretreatment with 3-AB resulted in slightly less enhancement at the low dose rate than at the high. Split-dose studies (9 Gy total dose) with radiation alone resulted in a recovery ratio of 1.4-1.5. Administration of 3-AB before the first dose resulted in a similar recovery ratio, but if given immediately after the first dose the ratio was smaller. Pretreatment of mice with the radiosensitizer RSU-1069 indicated that at least some of the stem cells were radiobiologically hypoxic. We suggest therefore that the enhancement of spermatogonial stem-cell killing by 3-AB is not entirely due to inhibition of repair processes but may also involve modification of the oxygen status of the testis.     

Response of mouse spermatogonial stem cells to X-ray induction of heritable reciprocal translocations

Although heritable translocations are an important endpoint for the assessment of genetic risk from radiation, there has been a serious information gap with regard to thier induction in spermatogonical stem cells, the most important cell stage in males for risk considerations. This led to uncertainty in estimating the magnitude of risk per unit exposure. Further, the relationship between the frequency of r eciprocal exchanges scored by cytological analysis of the exposed male's meiocytes and the frequency of those transmitted to first-generation offspring needed to be re-examined. In order to fill in these gaps, two radiation studies, i.e., dose response and dose fractionation, were conducted on spermatogonial stem cells in which heritable and cytologically detected translocations were scored.The present data are by far the most extensive, to date, for heritable translocation induction in spermatogonial stem cells. The linearity of the rising portion of the dose-effect curve and the additivity of effects observed in the fractionation study allow a direct estimation of the number of transmissible translocations expected per unit exposure. Thus,t he expected increase in heritable translocations per rad of acute X-rays in 3.89 × 10^?5 per gamete. The data also show a lack of consistensy between cytologically and genetically scored translocations.     

Inhibition of ADP-ribosylation increases X-ray-induced chromosomal damage in mouse testis and bone-marrow cells in vivo

The effect of 3-aminobenzamide (3AB) treatment on chromosomal radiosensitivity of mouse spermatogonial stem cells and bone-marrow cells was studied using various doses of X-rays. The results show that 3AB increases the induction of reciprocal translocations in slowly cycling spermatogonia as well as the frequency of chromosomal aberrations in actively dividing bone-marrow cells. The experiments indicate that both types of tissue are suitable to study the ability of inhibitors of ADP-ribosylation to modulate chromosome-breaking damage induced by ionizing radiation in vivo.     

X-ray-induced translocations in marmoset (Callithrix jacchus) stem-cell spermatogonia

The induction of reciprocal translocations in spermatogonial stem cells of marmosets (Callithrix jacchus) was studied after irradiation with different doses of X-rays (50, 100 and 200 rad) via spermatocyte analysis many cell generations later. The obtained results show a dose-effect relationship with clear saturation effects at 200 rad. The recorded frequencies of translocations were much lower than those reported for closely related marmosets (Saguinus fuscicollis and Saguinus oepidus). Possible reasons for this difference are discussed.     

Difference in the response of two hybrid stocks of mice to X-ray induction of chromosome aberrations in spermatogonial stem cells

Ionizing radiation induces balanced reciprocal translocations in spermatogonial stem cells of mice. From cells carrying these rearrangements, which can be scored cytologically in the diakinesis-metaphase I stage, balanced normal, balanced translocated and unbalanced (duplication/deficiency) sperm can be produced. The relationship between expected (calculated from cytological data) and observed frequencies of embryonic lethality (presumably as a result of unbalanced sperm fertilizing the egg) following exposure of spermatogonial stem cells to X-rays was studied in two hybrid stocks. A marked difference in the incidence of induced embryonic lethality was found between the two stocks. Similarly, a difference in the cytological frequencies of translocations was also found, although smaller than that observed for embryonic lethality. Thus, it appears that the difference between the two stocks in the frequencies of embryonic lethality may be attributable both to processes occurring prior to metaphase I and to a difference in the rate of transmission of unbalanced chromosome constitutions.     

Serial analysis of chromosomal aberrations and proliferation kinetics of mouse spermatogonia after single or fractionated X-ray exposures

Dose-fractionation studies on translocation induction in stem-cell spermatogonia of mice, as measured by spermatocyte analysis many cell generations after irradiation, revealed that a small conditioning dose of X-rays sensitizes the stem cells to the induction of translocations by a second dose 24 h later (Van Buul and Léonard, 1974, 1980). To find out whether such sensitization effects also occur at other spermatogonial stages, a comparison was made of the effects of single (50, 100 and 150 rad) and fractionated (100 + 50 rad, with 24 h in between) doses of X-rays on the induction of chromosomal aberrations in spermatogonia by analysing spermatogonial metaphases shortly after irradiation at multiple sampling times (0–48 h; every 4 h). In addition, the kinetics of spermatogonial proliferation was studied by using, in vivo, a BrdU chromosome-labelling procedure. The recorded frequencies of chromosomal aberrations did not indicate any sensitization effect of dose fractionation. It is concluded that the sensitization effects, as observed for chromosomal aberrations in male premeiotic germ cells, are characteristic for the stem-cell spermatogonia and do not occur in the more differentiated spermatogonia.     

Proliferative response of mouse spermatogonial stem cells after irradiation: a quantitative model analysis of experimental data

The testes of CDF₁ mice were irradiated with single doses of X-rays ranging from 2–16 Gy. The number of haploid cells in the testis at different times after irradiation (42–350 days) was determined by one-parameter flow cytometry both for irradiated animals and for age-matched controls. Based on literature data on the kinetics of the spermatogenesis in mice, a mathematical model of the (hierarchical) germ tissue was developed. Using this model, the processes of radiation-induced cell loss and subsequent recovery were simulated and free parameters of the model were estimated by fitting the model prediction to the experimental data. One of the aims of the study was to investigate the kinetic behaviour of spermatogonial stem cells and the corresponding control mechanisms. In order to fit the data, the model has to include the following features: (i) A preferential self-repopulation of spermatogonial stem cells following tissue injury. The model-estimated probability of a self-renewing division rises from 50% (the steady-state value) to 95% if the stem-cell population is reduced to 10% of its normal size. (ii) A relatively low, almost constant turnover rate of the stem-cell compartment. It is suggested by the analysis that less than 10% of the permatogonial stem cells present in the testis divide per day, regardless of the degree of cellular depletion. (iii) A mechanism responsible for incomplete recovery. The observed incomplete recovery of spermatogenesis after single doses exceeding 10 Gy can be described quantitatively assuming that the stem cells are organized into discrete proliferative structures, the number of cells per structure being about 60.     

Induction of specific locus mutations in mouse spermatogonial stem cells by combined chemical X-ray treatments

Data that demonstrate how the biology of spermatogenesis plays an important role in determining the yield of genetic damage from ionizing radiation are briefly reviewed. It is suggested that for valid extrapolations of data from mouse mutation experiments to man detailed knowledge of the spermatogonial stem cell systems in the two species is required. Two new sets of mouse specific mutation data are presented. (1) When a 2 mg/kg dose of triethylenemelamine (TEM) was used as a conditioning dose and followed 24 h later by 6 Gy X-rays, the mutation yield from spermatogonial stem cells was over twice as high (30.20 X 10(-5)/locus/gamete) as that when the X-ray dose was given alone (13.75 X 10(-5)/locus/gamete). No such effect was found when the TEM was given only 3 h prior to the X-irradiation. Since TEM at the dose used is inefficient at inducing specific-locus mutations, an augmentation of the X-ray response is indicated. It has therefore been concluded that the augmented mutation responses obtained with equal 24 h X-ray fractionations at high doses are attributable to mutation induction by the second dose. The responsive cells would be the formerly resistant component of the stem cell population that had survived the TEM treatment and that had been 'triggered' into a radiosensitive phase by the population depletion. (2) When 2 doses of 500 mg/kg hydroxyurea (HU) were given 3 h apart 3 h prior to 6 Gy X-rays to reduce the numbers of stem cells in the S and G2 phases of the cell cycle exposed to the radiation, the mutation responses was greatly enhanced to a level that is the highest yet recorded per unit X-ray dose (7.10 X 10(-5)/locus/gamete/Gy). No such effect was obtained when the intervals between the HU and X-ray treatments were either shorter (less than 0.5 h) or longer (24 h). It was concluded that X-ray-induced specific-locus mutations derive principally from stem cells in the G1 phase of the cell cycle. The reasons why the X-ray-induced mutation-yields from repopulating stem cells (with a short cell cycle and, hence, short G1 phase) are similar to those from undamaged stem cell populations, in contrast to translocation yields, therefore remains unresolved.     

The induction of reciprocal translocations in rhesus monkey stem-cell spermatogonia: effects of low doses and low dose rates

The induction of reciprocal translocation in rhesus monkey spermatogonial stem cells was studied following exposure to low doses of acute X rays (0.25 Gy, 300 mGy/min) or to low-dose-rate X rays (1 Gy, 2 mGy/min) and gamma rays (1 Gy, 0.2 mGy/min). The results obtained at 0.25 Gy of X rays fitted exactly the linear extrapolation down from the 0.5 and 1.0 Gy points obtained earlier. Extension of X-ray exposure reduced the yield of translocations similar to that in the mouse by about 50%. The reduction to 40% of translocation rate after chronic gamma exposure was clearly less than the value of about 80% reported for the mouse over the same range of dose rates. Differential cell killing with ensuing differential elimination of aberration-carrying cells is the most likely explanation for the differences between mouse and monkey.     

Dose-response studies on the spermatogonial stem cells of the rhesus monkey (Macaca mulatta) after X irradiation

Studies of the dose response of the spermatogonial stem cells in the rhesus monkey were performed at intervals of 130 and 160 days after graded doses of X irradiation. The D0 of the spermatogonial stem cells was established using the total numbers of the type A spermatogonia that were present at 130 and 160 days after irradiation and was found to be 1.07 Gy; the 95% confidence interval was 0.90-1.34 Gy.     

The radiosensitivity of spermatogonial stem cells in C3H/101 F1 hybrid mice

The radiosensitivity of spermatogonial stem cells of C3H/HeH × 101/H F₁ hybrid mice was determined by counting undifferentiated spermatogonia at 10 days after X-irradiation. During the spermatogenic cycle, differences in radiosensitivity were found, which were correlated with the proliferative activity of the spermatogonial stem cells. In stage VIII_irr, during quiescence, the spermatogonial stem cells were most radiosensitive with a D₀ of 1.4 Gy. In stages XI_irr−V_irr, when the cells were proliferatively active, the D₀ was about 2.6 Gy. Based on the D₀ values for sensitive and resistant spermatogonia and on the D₀ for the total population, a ratio of 45:55% of sensitive to resistant spermatogonial stem cells was estimated for cell killing.

When the present data were compared with data on translocation induction obtained in mice of the same genotype, a close fit was obtained when the translocation yield (Y; in % abnormal cells) after a radiation dose D was described by Y = e^τD, with τ = 1 for the sensitive and τ = 0.1 for the resistant spermatogonial stem cells, with a maximal e^τD of 100.     

Effects of hormone treatment on chromosomal radiosensitivity of somatic and germ cells of Snell's dwarf mice

The X-ray induction of micronuclei and structural chromosomal aberrations was studied in bone-marrow cells of normal and dwarf (dw) mice in combination with thyroxine and/or prolactin treatment or otherwise. Hormone treatment clearly increased micronuclei induction but not chromosome breakage, suggesting that indirect effects were involved. Since no clear differences in the timing of the final stage of erythropoiesis could be found, it is likely that the indirect effects are mediated via the formation-differentiation kinetics of erythroblasts. The induction of reciprocal translocations by X-rays in stem cell spermatogonia of dwarf mice was lower than in normals and treatment with prolactin, growth hormone and/or thyroxin, did not influence the chromosomal radiosensitivity of spermatogonial stem cells.