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.     

Nonrandom distribution of mouse spermatogonial stem cells surviving fission neutron irradiation

Colony formation by surviving spermatogonial stem cells was investigated by mapping pieces of whole mounted tubuli at intervals of 6 and 10 days after doses of 0.75 and 1.50 Gy of fission neutron irradiation. Colony sizes, expressed in numbers of spermatogonia per colony, varied greatly. However, the mean colony size found in different animals was relatively constant. The mitotic indices in large and small colonies and in colonies in different epithelial stages did not differ significantly. This finding suggests that size differences in these spermatogenic colonies are not caused by differences in growth rate. Apparently, surviving stem cells start to form colonies at variable times after irradiation. The number of colonies per unit area varied with the epithelial stages. Many more colonies were found in areas that during irradiation were in stages IX-III (IX-IIIirr) than in those that were in stages IV-VII (IV-VIIirr). After a dose of 1.50 Gy, 90% of all colonies were found in areas IX-IIIirr. It is concluded that the previously found difference in repopulation after irradiation between areas VIII-IIIirr and III-VIIIirr can be explained not by differences in colony sizes and/or growth rates of the colonies in these areas but by a difference in the number of surviving stem cells in both areas. In area XII-IIIirr three times more colonies were found after a dose of 0.75 Gy than after a dose of 1.50 Gy. In area IV-VIIirr the numbers of colonies differed by a factor of six after both doses. This finding indicates that spermatogonial stem cells are more sensitive to irradiation in epithelial stages IV-VII than in stages XII-III. In control material, spermatogonia with a nuclear area of 70-110 micron2 are rare. However, especially 6 days after irradiation, single cells of these dimensions are rather common. These cells were found to lie at random over the tubular basement membrane with no preference for areas with colonies. It is concluded that the great majority of these cells were not or do not derive from surviving stem cells. These enlarged cells most likely represent lethally injured cells that will die or become giant cells (nuclear area greater than 110 micron2).     

A critical stage in spermatogenesis for radiation-induced cell death in the medaka fish, Oryzias latipes

To ensure the high-fidelity transmission by reproductive cells of genetic information from generation to generation, cells have evolved surveillance systems to eliminate genomic lesions by inducing cell suicide and/or DNA repair. In this report, gamma-ray-induced cell death was investigated using the medaka fish, Oryzias latipes, because of the ease with which the differentiation stages of its spermatogenic cells can be identified. After 4.75 Gy gamma irradiation, the maximum rate of death of spermatogonial stem cells was observed at 18 h, and that of differentiating spermatogonia was at 12 h, followed by a peak in the extent of DNA fragmentation detected by the TUNEL assay. Dose-response curves for the death rate showed an obvious increase in the death rate for early-differentiating spermatogonia even after 0.11 Gy irradiation, whereas there were no such increases for spermatogonial stem cells and late-differentiating spermatogonia. In the male germ cells of this fish, the stage during spermatogenesis most sensitive to radiation-induced cell death is in early-differentiating spermatogonia, the immediate descendants of the stem cells. These spermatogonia may have a rigorous surveillance system for genomic lesions induced in spermatogonial stem cells.     

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.     

The sensitivity of quiescent and proliferating mouse spermatogonial stem cells to X irradiation.

The radiosensitivity of spermatogonial stem cells to X rays was determined in the various stages of the cycle of the seminiferous epithelium of the CBA mouse. The numbers of undifferentiated spermatogonia present 10 days after graded doses of X rays (0.5-8.0 Gy) were taken as a measure of stem cell survival. Dose-response relationships were generated for each stage of the epithelial cycle by counting spermatogonial numbers and also by using the repopulation index method. Spermatogonial stem cells were found to be most sensitive to X rays during quiescence (stages IV-VII) and most resistant during active proliferation (stages IX-II). The D0 for X rays varied from 1.0 Gy for quiescent spermatogonial stem cells to 2.4 Gy for actively proliferating stem cells. In most epithelial stages the dose-response curves showed no shoulder in the low-dose region.     

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.     

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.     

Efficiency of adult mouse spermatogonial stem cell colony formation under several culture conditions

The aim of this study was to compare the in vitro effects of glial cell line-derived neurotrophic factor, stem cell factor, granulocyte macrophage-colony stimulating factor, and co-culture with Sertoli cells on the efficiency of adult mouse spermatogonial stem cells colony formation. For these purpose, both Sertoli and spermatogonial cells were isolated from adult mouse testes. The identity of the cells was confirmed through analysis of alkaline phosphatase activity, immunocytochemistry against OCT-4, c-kit, and vimentin, and also by transplantation of these cells in the recipient testes. The isolated spermatogonial cells were treated either with various concentrations of the above mentioned factors or co-cultured with Sertoli cells for 3 wk. The spermatogonial cells of the resulting colonies were transplanted via rete testis into the mouse testes, which were irradiated with 14 Gy. The results indicated that glial cell line-derived neurotrophic factor is the most appropriate factor for in vitro colonization of adult mice spermatogonial cells compared with other cytokines and growth factors. A short-term co-culture with Sertoli cells showed a significant increase in the number and diameter of the colonies compared with the treated growth factors and the control group. We have also demonstrated that mouse spermatogonial stem cells in the colonies after co-culturing with Sertoli cells could induce spermatogenesis in the recipient testes after transplantation.     

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.     

Effect of irradiation on cytokine secretion and nitric oxide production by inflammatory macrophages

This study explored the effects of low-dose and high-dose irradiation on inflammatory macrophage cells, specifically inflammatory cytokine secretion and nitric oxide (NO) production after irradiation. To elucidate the effect of irradiation on active and inactive macrophages, we exposed LPS-treated or untreated murine monocyte/macrophage RAW 264.7 cell lines to low-dose to high-dose radiation (0.01–10 Gy). We analyzed the effects of irradiation on RAW 264.7 cell proliferation by MTT assays and analyzed cytokine secretion and NO production related to inflammation by ELISA assays. Low-to-high doses of radiation did not significantly affect the proliferation of LPS-treated or untreated RAW 264.7 cells. Pro-inflammatory cytokine IL-1ß was generally increased in RAW 264.7 cells at 3 days after radiation. Especially, IL-1ß was significantly increased in only high dose-irradiation (2 and 10 Gy irradiation) groups in LPS-untreated RAW 264.7 cells but increased in both low and high dose-irradiation groups (0.01–10 Gy) in LPS-treated RAW 264.7 cells at 3 days after irradiation. Whereas, the expression of IL-1ß was prolonged in high-dose irradiation group at 5 days after irradiation. The production of anti-inflammatory cytokine IL-10 did not change significantly at 3 days after radiation but was significantly reduced at 5 days after 10 Gy radiation. The effect of irradiation on the secretion of IL-1ß and IL-10 was not significantly different between RAW 264.7 cells treated or not treated with LPS. The effect of irradiation on NO secretion by RAW 264.7 cells showed a specific pattern. NO was produced after low-dose irradiation but reduced in a high-dose irradiation group at 3 days after irradiation. However, NO production was not changed after low-dose irradiation and reduced at 5 days after high-dose irradiation. These results showed that irradiation affected the inflammatory system and regulated NO production in both activated and inactivated macrophages through different regulation mechanisms, depending on irradiation dose.     

Variation in the sensitivity of the mouse spermatogonial stem cell population to fission neutron irradiation during the cycle of the seminiferous epithelium

Dose-response studies of the radiosensitivity of spermatogonial stem cells in various epithelial stages after irradiation with graded doses of fission neutrons of 1 MeV mean energy were carried out in the Cpb-N mouse. These studies on the stem cell population in stages IX-XI yielded simple exponential lines characterized by an average D0 value of 0.76 +/- 0.02 Gy. In the subsequent epithelial stages XII-III, a significantly lower D0 value of 0.55 +/- 0.02 Gy was found. In contrast to the curves obtained for stem cells in stages IX-III, the curves obtained in stages IV-VIII indicated the presence of a mixture of radioresistant and radiosensitive stem cells. In stage VII, almost no radioresistant stem cells appeared to be present and a D0 value for the radiosensitive stem cells of 0.22 +/- 0.01 Gy was derived. Previously, data were obtained on the size of colonies (in number of spermatogonia) derived from surviving stem cells. Combining these data with data from the newly obtained dose-response curves yielded the number of stem cells, per stage and with the specific radiosensitivities, present in the control epithelium. In stages IX-XI, there are approximately 6 stem cells per 1000 Sertoli cells with a radiosensitivity characterized by a D0 of 0.76 Gy, which corresponds to one-third of the As population in these stages. (The As spermatogonia are presumed to be the stem cells of spermatogenesis.) IN stages XII-III, there are approximately 12 stem cells per 1000 Sertoli cells with a radiosensitivity characterized by a D0 of 0.55 Gy, which roughly equals the number of A single spermatogonia in these stages. These calculations could not be made for stages IV-VIII since no simple exponential lines were obtained for these stages. In view of the pattern of the proliferative activity of the spermatogonial stem cells during the epithelial cycle, it appears that the stem cell population is most radiosensitive during the period when the majority of these cells are in G0 phase, most resistant when the cells are stimulated again into proliferation, and of intermediate sensitivity during active proliferation.     

Long-term effects of irradiation before adulthood on reproductive function in the male rhesus monkey.

Today, many patients, who are often young, undergo total body irradiation (TBI) followed by bone marrow transplantation. This procedure can have serious consequences for fertility, but the long-term intratesticular effects of this treatment in primates have not yet been studied. Testes and epididymides of rhesus monkeys that received doses of 4-8.5 Gy of TBI at 2-4 yr of age were studied 3-8 yr after irradiation. In all irradiated monkeys, at least some seminiferous tubule cross-sections lacked germ cells, indicating extensive stem cell killing that was not completely repaired by enhanced stem cell renewal, even after many years. Testes totally devoid of germ cells were only found in monkeys receiving doses of 8 Gy or higher and in both monkeys that received two fractions of 6 Gy each. By correlating the percentage of repopulated tubules (repopulation index) with testicular weight, it could be deduced that considerable numbers of proliferating immature Sertoli cells were killed by the irradiation. Because of their finite period of proliferation, Sertoli cell numbers did not recover, and potential adult testis size decreased from approximately 23 to 13 g. Most testes showed some dilated seminiferous tubules, indicating obstructed flow of the tubular fluid at some time after irradiation. Also, in 8 of the 29 irradiated monkeys, aberrant, densely packed Sertoli cells were found. The irradiation did not induce stable chromosomal translocations in spermatogonial stem cells. No apparent changes were seen in the epididymides of the irradiated monkeys, and the size of the epididymis adjusted itself to the size of the testis. In the irradiated monkeys, testosterone and estradiol levels were normal, whereas FSH levels were higher and inhibin levels lower when testicular weight and spermatogenic repopulation were low. It is concluded that irradiation before adulthood has considerable long-term effects on the testis. Potential testis size is reduced, repopulation of the seminiferous epithelium is generally not complete, and aberrant Sertoli cells and dilated tubules are formed. The latter two phenomena may have further consequences at still longer intervals after irradiation.     

Gamma-ray exposure accelerates spermatogenesis of medaka fish,Oryzias latipes

To examine the spermatogenesis (and spermiogenesis) cell population kinetics after gamma-irradiation, the frequency and fate of BrdU-labeled pre-meiotic spermatogenic cells (spermatogonia and pre-leptotene spermatocytes) and spermatogonial stem cells (SSCs) of the medaka fish (Oryzias latipes) were examined immunohistochemically and by BrdU-labeling. After 4.75 Gy of gamma-irradiation, a statistically significant decrease in the frequency of BrdU-labeled cells was detected in the SSCs, but not in pre-meiotic spermatogenic cells. The time necessary for differentiation of surviving pre-meiotic spermatogenic cells without delay of germ cell development was shortened. More than 90% of surviving pre-meiotic spermatogenic cells differentiated into haploid cells within 5 days after irradiation, followed by a temporal spermatozoa exhaust in the testis. Next, spermatogenesis began in the surviving SSCs. However, the outcome was abnormal spermatozoa, indicating that accelerated maturation process led to morphological abnormalities. Moreover, 35% of the morphologically normal spermatozoa were dead at day 6. Based on these results, we suggest a reset system; after irradiation most surviving spermatogenic cells, except for the SSCs, are prematurely eliminated from the testis by spermatogenesis (and spermiogenesis) acceleration, and subsequent spermatogenesis begins with the surviving SSCs, a possible safeguard against male germ cell mutagenesis.     

Modification of radiation-induced oral mucositis (mouse) by adult stem cell therapy: single-dose irradiation

Early oral mucositis occurs in response to accidental upper partial body exposure as well as to radiotherapy in the head-and-neck region. This study was initiated to define the potential of mobilization of endogenous bone marrow (BM) stem cells by rHuG-CSF or of bone marrow transplantation (BMT) to reduce the effect of single-dose irradiation on mouse oral epithelium. A 3 × 3 mm² area of the lower tongue surface of mice was irradiated with graded single doses (day 0). Mucosal ulceration was used as the endpoint for dose–response analyses. Stem cells were mobilized by rHuG-CSF (8 times/4 days), timed to achieve a maximum of circulating stem cells on days 0, +1, +4, +8 or +10. Alternatively, syngeneic BM was transplanted on these days. The ED₅₀ (dose at which ulceration is expected in 50 % of the animals) for irradiation alone was 11.9 ± 3.4 Gy. Mobilization of stem cells with a maximum of circulating stem cells on days +4, +8 or +10 significantly increased the ED₅₀ to 25.5 ± 10.1, 23.5 ± 10.1 and 26.5 ± 13.0 Gy. In contrast, a maximum of circulating stem cells on day 0 or day +1 had no effect. BMT did not result in a significant change in isoeffective doses in any of the protocols. In conclusion, the response of oral mucosal epithelium to a single-radiation exposure can be significantly reduced by post-exposure mobilization, but not by transplantation, of BM stem cells.     

The role of the tumor suppressor p53 in spermatogenesis

The p53 protein appeared to be involved in both spermatogonial cell proliferation and radiation response. During normal spermatogenesis in the mouse, spermatogonia do not express p53, as analyzed by immunohistochemistry. However, after a dose of 4 Gy of X-rays, a distinct p53 staining was present in spermatogonia, suggesting that, in contrast to other reports, p53 does have a role in spermatogonia. To determine the possible role of p53 in spermatogonia, histological analysis was performed in testes of both p53 knock out C57BL/6 and FvB mice. The results indicate that p53 is an important factor in normal spermatogonial cell production as well as in the regulation of apoptosis after DNA damage. First, p53 knock out mouse testes contained about 50% higher numbers of A1 spermatogonia, indicating that the production of differentiating type spermatogonia by the undifferentiated spermatogonia is enhanced in these mice. Second, 10 days after a dose of 5 Gy of X-rays, in the p53 knock out testes, increased numbers of giant sized spermatogonial stem cells were found, indicating disturbance of the apoptotic process in these cells. Third, in the p53 knock out testis, the differentiating A2-B spermatogonia are more radioresistant compared to their wild-type controls, indicating that p53 is partly indispensable in the removal of lethally irradiated differentiating type spermatogonia. In accordance with our immunohistochemical data, Western analysis showed that levels of p53 are increased in total adult testis lysates after irradiation. These data show that p53 is important in the regulation of cell production during normal spermatogenesis either by regulation of cell proliferation or, more likely, by regulating the apoptotic process in spermatogonia. Furthermore, after irradiation, p53 is important in the removal of lethally damaged spermatogonia.     

Dose-effect relationship for X-ray-induced reciprocal translocations in mouse spermatogonia following pretreatment with 3-aminobenzamide

The influence of 3-aminobenzamide (3-AB) pretreatment on the dose-response relationship for radiation-induced reciprocal translocations in mouse spermatogonial stem cells was studied. The results show that at doses of 3-10 Gy of X-rays the frequencies of translocations were higher in 3-AB-pretreated animals as compared to animals that received X-rays only. The 3-AB pretreatment was not effective at dose levels of 1 and 2 Gy. The shape of the dose-effect curve was similar to that obtained without 3-AB pretreatment, i.e., a humped curve, but the initial slope was clearly steeper and the position of the peak was shifted from 7 to 9 Gy. The effects observed can be explained by a 3-AB-mediated sensitization of normally radioresistant stem cells that are at the stage of stimulation to enter the mitotic cycle, thus increasing the population of radiosensitive spermatogonial stem cells.     

Increased number of cardiac adrenergic receptors following local heart irradiation

The effect of local X irradiation on cardiac alpha and beta receptors was studied in Wistar rats. Animals were given local heart irradiation with single doses of 15 or 20 Gy and were examined after a range of latency times of 7 to 400 days. Using the radioactive ligands [3H]CGP-12177 and [3H]prazosin, the maximal binding capacity was determined from saturation experiments. At 7 days after 20 Gy the maximal binding capacity of both alpha and beta receptors was reduced to below the level of untreated control animals. Subsequently it rose continually to a maximum of 160% of the control level for beta receptors and 130% for alpha receptors at 400 days postirradiation. The antagonist affinity as judged from the dissociation constant for [3H]CGP 12177 and [3H]prazosin did not change significantly. A similar effect was observed after 15 Gy. An increase in adrenergic receptors may represent an important pathogenetic link between early morphological and late functional changes in the pathogenesis of radiation-induced heart disease.     

Induction of congenital malformations in the offspring of male mice treated with X-rays at pre-meiotic and post-meiotic stages

The induction of congenital malformations among the offspring of male mice treated with X-rays at pre-meiotic and post-meiotic stages has been studied in two experiments. Firstly, animals were exposed to varying doses (108–504 cGy) of X-rays and mated at various time intervals (1–7, 8–14, 15–21 and 64–80 days post-irradiation), so as to sample spermatozoa, spermatids and spermatogonial stem cells. In the second experiment, only treated spermatogonial stem cells were sampled. One group of males was given a single 500-cGy dose, a second group a fractionated dose (500 + 500 cGy, 24 h apart) and a third group was left unexposed.In the first experiment, induced post-implantation dominant lethality increased with dose, and was highest in week 3, in line with the known greater radiosensitivity of the early spermatid stage. Preimplantation loss also increased with dose and was highest in week 3. There was no clear induction of either pre-implantation or post-implantation loss at spermatogonial stem cell stages.There was a clear induction of congenital malformations at post-meiotic stages, the overall incidence being 2.0 ± 0.32% in the irradiated series and 0.24 ± 0.17% among the controls. The induction was statistically significant at each dose. At the two highest doses the early spermatids (15–21 days) appeared more sensitive than spermatozoa, and at this stage the incidence of malformations increased with dose. The data from Expt. 1 on the induction of malformations by irradiation of spermatogonial stages were equivocal. In contrast, Expt. 2 showed a statistically significant induction of malformations at both dose levels (2.2 ± 0.46% after 500 cGy and 3.1 ± 0.57% after 500 + 500 cGy). The relative sensitivities of male stem cells, post-neiotic stages and mature oocytes to the induction of congenital malformations were reasonably similar to their sensitivities for specific-locus mutations, except that the expected enhancing effect of the fractionation regime used was not seen.Dwarfism and exencephaly were the two most commonly observed malformations in all series.     

Induction of external abnormalities in offspring of male mice irradiated with 252Cf neutron.

To assess the genetic effects of fission neutron, the induction of external malformations was studied in F1 fetuses after F0 male mice were irradiated. Male mice of the ICR:MCH strain were irradiated with 252Cf neutron at doses of 0.238, 0.475, 0.95 and 1.9 Gy. They were mated with non-irradiated female mice at 71-120 days after the irradiation. Pregnant females were autopsied on day 18 of gestation and their fetuses were examined for deaths and external abnormalities. No increases of pre- and post-implantation losses were noted at any dose. External abnormalities were observed at rates of 1.40% in the 0.238 Gy, 2.23% in the 0.475 Gy, 3.36% in the 0.95 Gy and 3.26% in the 1.9 Gy groups; the rate in the control group was 1.65%. The dose-response curve was linear up to 0.95 Gy, and then flattened out; the induction rate of external abnormalities was 2.7 x 10(-4)/gamete/cGy based on the linear regression. These results indicated that fission neutron effectively induces external abnormalities in F1 fetuses after spermatogonial irradiation.     

Modification of radiation myelopathy by the transplantation of neural stem cells in the rat

In a novel approach, neural stem cells were transplanted to ameliorate radiation-induced myelopathy in the spinal cords of rats. A 12-mm section of the cervical spinal cord (T2-C2) of 5-week-old female Sprague-Dawley rats was locally irradiated with a single dose of 22 Gy of (60)Co gamma rays. This dose is known to produce myelopathy in all animals within 6 months of irradiation. After irradiation, the animals were subdivided into three groups, and at 90 days after irradiation, neural stem cells or saline (for controls) were injected into the spinal cord, intramedullary, at two sites positioned 6 mm apart on either side of the center of the irradiated length of spinal cord. The injection volume was 2 microl. Group I received a suspension of MHP36 cells, Group II MHP15 cells, and Group III (controls) two injections of 2 microl saline. All rats received 10 mg/kg cyclosporin (10 mg/ml) daily i.p. to produce immunosuppression. All animals that received saline (Group III) developed paralysis within 167 days of irradiation. The paralysis-free survival rates of rats that received transplanted MHP36 and MHP15 cells (Groups I and II) were 36.4% and 32% at 183 days, respectively. It was concluded that transplantation of neural stem cells 90 days after irradiation significantly (P = 0.03) ameliorated the expression of radiation-induced myelopathy in the spinal cords of rats.