Comparison of frequencies of radiation-induced stable chromosomal aberrations in somatic and germ tissues of the mouse

Radiation-induced stable chromosome aberrations have been studied in the testes and bone-marrow of the mouse (Mus musculus). 60 days after whole-body irradiation with a dose of 400 rad X-rays, the frequency of visible chromosome aberrations in bone-marrow cells was 19.8%. The frequency of chromosome aberrations in spermatogonia of the same mice, scored as multivalents in spermatocytes, was considerably lower — only 4.7%. The possible mechanisms underlying this marked difference in translocation yield are discussed.     

Griseofulvin-induced aneuploidy and meiotic delay in male mouse germ cells: detected by using conventional cytogenetics and three-color FISH.

Griseofulvin (GF) was tested in male mouse germ cells for the induction of meiotic delay and aneuploidy. Starved mice were orally treated with 500, 1000 and 2000 mg/kg of GF in corn oil and testes were sampled 22 h later for meiotic delay analysis and chromosome counting in spermatocytes at the second meiotic metaphase (MMII). A dose-related increase in meiotic delay by dose-dependently arresting spermatocytes in first meiotic metaphase (MMI) or/and prolonging interkinesis was observed. Hyperhaploid MMII cells were not significantly increased. Sperm were sampled from the Caudae epididymes 22 days after GF-treatment of the males for three-color fluorescence in situ hybridization (FISH). The frequencies of diploidies were 0.01-0.02% in sperm of the solvent control animals and increased dose-dependently to 0.03%, 0.068% and 0.091%, respectively, for 500, 1000 and 2000 mg/kg of GF. The frequencies of disomic sperm were increased significantly above the controls in all GF-treated groups but showed no dose response. The data for individual classes of disomic sperm indicated that MII was more sensitive than MI to GF-induced non-disjunction in male mice. A comparison of the present data from male mice and literature data from female mice suggests that mouse oocytes are more sensitive than mouse spermatocytes to GF-induced meiotic delay and aneuploidy.     

Clastogenic effects of hydroquinone: induction of chromosomal aberrations in mouse germ cells

The clastogenic activity of hydroquinone (HQ) in germ cells of male mice was evaluated by analysis of chromosomal aberrations in primary spermatocytes and differentiating spermatogonia. In the first experiment with treated spermatocytes the most sensitive stage of meiotic prophase to aberration induction by HQ was determined. Testicular material was sampled for microscopic analysis of cells in diakinesis-metaphase I at 1, 5, 9, 11, and 12 days after treatment with 80 mg/kg of HQ, corresponding to treated diplotene, pachytene, zygotene, leptotene and preleptotene. The frequencies of cells with structural chromosome aberrations peaked at 12 days after treatment (p less than 0.01). This indicates that the preleptotene when DNA synthesis occurred was the most sensitive stage of meiotic prophase. In the second experiment the dose response was determined 12 days post treatment by applying 2 additional doses of 40 mg/kg and 120 mg/kg. The clastogenic effects induced by 40 and 80 mg/kg were significantly different from the controls (p less than or equal to 0.01) and higher than the results obtained with 120 mg/kg of HQ. A humped dose-effect relationship was observed. In a third experiment the same doses were used to analyse chromosomal aberrations in dividing spermatogonia of mice 24 h after treatment with HQ. All the administered doses gave results statistically different from the control values (p less than or equal to 0.01) and the data were fitted to a linear equation. HQ was found to be clastogenic in male mouse germ cells. It is concluded that the clastogenic effect in male germ cells is of the same order of magnitude as in mouse bone marrow cells.     

Cytogenetic effects of malathion insecticide on somatic and germ cells of mice

Male mice dermally exposed to single or multiple treatment (5 days/2 weeks) showed that the ability of malathion to induce chromosome aberrations in somatic (bone marrow) and germ cells (primary spermatocytes) was related to the type of treatment and dose used. Statistically significant increases of chromosome aberrations in bone marrow cells occurred after single treatment (500 and 2000 mg/kg body wt) when chromatid gaps were included and after multiple treatment (250 and 500 mg/kg) when they were excluded. No dose-response relationships were observed for either treatment. In germ cells, malathion induced a significant increase of univalents in both types of treatment but structural chromosome aberrations were induced only by multiple treatment. Malathion induced a significant decrease of the mitotic indices in the bone marrow.     

A comparative study of the frequencies of radiation-induced chromosome aberrations in somatic and germ cells of the rhesus monkey (Macaca mulatta)

Frequencies of radiation-induced chromosome aberrations in spermatogonia, peripheral blood lymphocytes and bone-marrow cells of the rhesus monkey (Macaca mulatta) and in human blood lymphocytes, were determined at different exposures of X-rays. The dose-response curve for the induction of reciprocal translocations in spermatogonia suggested a “hump” at about 200 rad. The absolute frequencies of chromosome aberrations in somatic and germ cells of the rhesus monkey were low in comparison with most other mammalian species and the ratio between aberrations in the two tissues was 25 to 1 at the 100 rad level. Although the numbers of “effective chromosome arms” in man and rhesus monkey are similar (81 vs. 83), the rhesus monkey showed a lower rate of induction of dicentrics in blood lymphocytes than man at all doses, reaching statistical significance at the 300 rad level.     

Cytogenetic effects of X-rays and fission neutrons in female mice

The induction by X-rays of chromosomal damage in oocytes was studied, while the genetic consequences of X- and neutron-induced damage in female mice were looked for by testing offspring for dominant lethality and semi-sterility. None out of 386 sons of hybrid females given 300 rad X-rays showed evidence of semi-sterility or translocation heterozygosity, but 9 out of 294 daughters were diagnosed as semi-sterile. At least 3 and probably 4 of these (1.4%) carried reciprocal translocations, 2 of which caused male sterility. Complete or partial loss of the X-chromosome may have been responsible for some of the other sermi-steriles. Examination of oocytes at metaphase-I during the first and third weeks after X-irradiation with 100 or 400 rad revealed both multivalents (some of the ring quadrivalent type) and fragments (mainly double). These were thought to arise mainly from chromatid intercchanges (both symmetrical and asymmetrical) and isochromatid intrachanges respectively. Since neither the proportion of asymmetrical interchanges nor the amount of hidden damage was known it was not thought possible to predict the magnitude of F₁ effects from metaphase-I findings. The aberration frequency in oocytes rose with dose and (at the 400 rad level only) with time after irradiation, reaching a maximum of 10% multivalents and 22% fragments in the third week after 400 rad. The frequency of univalents showed no consistent trend, but chiasma counts decreased in the first week after 400 rad. The increase in levels of chromosomal damage with dose and time after irradiation was reflected in dominant lethal frequencies after the same radiation-conception intervals and doses of 0–400 rad. Induced post-implantation lethality was over twice as high in the third week after 200–400 rad than in the first. Pre-implantation loss also greatly increased in the third week after 300 or 400 rad; this was associated with increased non-fertilization of ova. No evidence for the induction of translocations in oogonia or resting oocytes by fast neutron irradiation was obtained, although there was evidence for X-chromosomal loss after 200 rad to oocytes. The relative biological effectiveness (RBE) for fission neutrons vs. X-rays with respect to dominant lethal induction in oocytes was found to vary with dose, but seamed to be around 1 at lower levels.     

Radiation-induced and transposon-induced chromosome damage in Drosophila: translocations and transmission distortion

The possible interaction between X-ray- and transposon-induced chromosome damage was monitored in the P-M system of hybrid dysgenesis in Drosophila melanogaster. One- to two-day-old F1 dysgenic males originating from a cross between M strain females and P strain males were irradiated with 5.5 Gy (550 rad) or used as controls to monitor X-Y translocations and transmission ratio distortion. Two 3-day sperm broods were sampled for the former and two 4-day broods for the latter to detect damage induced in the most radiosensitive cells. F1 nondysgenic males derived from M female to M male crosses (controls) were treated identically. X-Y chromosome translocations induced by P element mobility alone declined sharply with a decrease in temperature (18 versus 21 degrees C) and they were significantly reduced with aging of hybrid males from brood 2, 4-8 days of age, to brood 3, 7-11 days of age. No significant increase in translocations was observed when X irradiation and P-M dysgenesis were combined, showing no interaction between damages induced by the two mutator systems. In contrast, interaction was observed in transmission ratio distortion which was significantly increased by X irradiation of hybrid males derived from both reciprocal M X P and P x M crosses. The preferential elimination of P element-bearing autosomes occurred when either spermatocytes or spermatids were irradiated. An aging effect was also observed, resulting in less distortion in 9- to 14-day-old dysgenic males compared to 5- to 10-day-old hybrids.     

X-ray-induced mutation spectra of different germ-cell stages of Drosophila males with a double marked Y-chromosome and either a normal X-or a ring-X-chromosome (author's transl)

Different germ-cell stages of Drosophila males with a double marked Y-chromosome and either a normal X- or a ring-X chromosome were irradiated with X-rays, inducing the following aberrations: chromosome loss, chromosome gain (XYX-females), partial Y loss and isochromosomes of the Y-chromosome.Doses of 520 rad in spermatocytes and spermatids and 2600 rad in sperm, produced the same effect in these stages with regard to the chromosome loss in the males with a normal X, and the following results were obtained: (a) The partial Y loss in postmeiotic stages is small in comparison with spermatocytes in both stocks. This could mean that in spermatocytes this aberration is determined by exchange processes which can only be induced and/or detected in premeiotic stages. (b) In spermatocytes and mature sperm of males with a ring-X chromosome, the chromosome loss was 2.9 times greater than in those with a normal X. In spermatids of the males with a ring-X the rate of loss was only 1.5 times greater. In spermatocytes of either males with a ring-X or a normal X a similar high rate of isochromosomes could be induced. However, in spermatids and mature sperm the rate of induction of isochromosomes was found to be very small. These results seem to indicate that in mature sperm the rejoining of breaks in the Y-chromosome takes place before, and in the X-chromosome usually after the replication. If in post-meiotic stages of Drosophila the X- and Y-chromosomes existed as chromatid-like subunits then in spermatids these should behave as a structural unit.In sperm we were able to induce similar frequencies of individuals with a single isochromosome type in all body cells as of individuals with two types of isochromosomes (isochromosome mosaics). This result seems to indicate that after irradiation of sperm one of the first two division nuclei is lethal in an proportion of the zygotes.     

Chromosome translocations in the sex cells of mice subjected to chronic low-dose gamma-irradiation

Translocation induction in mouse spermatogonia by continuous whole-body gamma irradiation (radium 226) was studied. Total doses, delivered at a rate of 13.0 +/- 1.3 X 10(-4) rad/min for various time intervals, were 97, 195, 294 and 442 rad. Cytological examination within 3 to 4 months after irradiation indicated the presence of translocations in 0.16, 0.30, 0.75 and 1.29 percent respectively, of primary spermatocytes at diakinesis metaphase I. Data on translocation induction (Y) as related to total irradiation dose (D) were best fitted to a second power parabola equation (Y=5.1 X 10(-6)D2 + 7.32 X 10(-4) X D). The results obtained confirm that chronic gamma irradiation is of low genetic efficiency, and support the suggestion that there exists a dose-rate threshold under which no more changes in exposure efficiency will occur.     

Dose—response relationship for X-ray-induced reciprocal translocations in stem-cell spermatogonia of the rhesus monkey (Macaca mulatta)

The induction of reciprocal translocation in stem-cell spermatogonia of the rhesus monkey (Macaca mulatta) was studied after testicular X-irradiation of mature males (50, 100 and 200 rad) or whole-body irradiation of young males (200 and 300 rad). After the recovery of the germinal epithelium, cytogenetic analysis was carried out on spermatocytes descended from irradiation spermatogonia. Preparations of C-banded diakinesis-metaphase I were screened for translocation configurations. The frequencies of abberations obtained were 0% at 0 rad, 0.36% at 50% rad, 0.86% at 100 rad, 0.99% at 200 rad and 0.68% at 300 rad, suggesting a humped dose—response relationship. There was no evidence for the contribution of a quadratic component to the yield in the lower dose range. A comparison of these results with those obtained for other mammals by a number on investigators shows that the frequencies of translocations in the rhesus monkey are much lower than those published for most other mammalian species.     

Cytogenetic effects of protracted gamma exposures from conception of male mice

In order to gain an overall picture of the genetic effects of an increased level of background radiation it is necessary to study the results of protracted exposures to embryonic and immature germ-cell stages as well as to stages found in the mature organism. For this purpose, litters produced by female mice, kept in a 10 or 20 rad/day ⁶⁰Co-γ-irradiation field, were kept in the same fields from conception until about 60 days later, having absorbed doses of 526 and 1078 rad respectively. Tests on exposed female offspring showed them to be sterile. 8 weeks after removal from the gamma field, mean testis masses of males in the 20 rad/day series were only half normal but those receiving 10 rad/day were little affected. Frequencies of translocations in spermatocytes at diakinesis/metaphase I were only slightly increased in the exposed series, differences not being significant. Estimated rates of translocation induction were around 5 × 10^?6 per rad, about one-third of those found after protracted γ-irradiation of stem-cell spermatogonia in the adult. Embryonic lethality in progeny of other similarly irradiated males (absorbed doses of 560 and 1040 rad), mated 2 months after removal from the radiation fields, was also increased slightly, but not significantly. Results are compared with others on the induction of chromosome aberrations and gene mutations, mainly by acute irradiation, in prenatal and neonatal male mice. It is concluded that early male germ-cell stages generally show a reduced genetic radiosensitivity after both acute and chronic exposures.     

Protection from radiation-induced male germ cell loss by sphingosine-1-phosphate

Male germ cells are susceptible to radiation-induced injury, and infertility is a common problem after total-body irradiation. Here we investigated, first, the effects of irradiation on germ cells in mouse testis and, second, the role of sphingosine-1-phosphate (S1P) treatment in radiation-induced male germ cell loss. Irradiation of mouse testes mainly damaged the early developmental stages of spermatogonia. The damage was seen by means of DNA flow cytometry 21 days after irradiation as decreasing numbers of spermatocytes and spermatids with increasing amounts of ionizing radiation (0.1-2.0 Gy). Intratesticular injections of S1P given 1-2 h before irradiation (0.5 Gy) did not protect against short-term germ cell loss as measured by in situ end labeling of DNA fragmentation 16 h after irradiation. However, after 21 days, in the S1P-treated testes, the numbers of primary spermatocytes and spermatogonia at G2 (4C peak as measured by flow cytometry) were higher at all stages of spermatogenesis compared with vehicle-treated testes, indicating protection of early spermatogonia by S1P, whereas the spermatid (1C) populations were similar. In conclusion, S1P appears to protect partially (16%-47%) testicular germ cells against radiation-induced cell death. This warrants further studies aimed at development of therapeutic agents capable of blocking sphingomyelin-induced pathways of germ cell loss.     

Differentiation of Primary Spermatocytes to Elongated Spermatids by Mammalian FSH in Organ Culture of Testes Fragments from the Newt, Cynops pyrrhogaster

Our previous studies (10, 11) showed that mammalian follicle-stimulating hormone (FSH) alone was indispensable and sufficient for the initiation and promotion of spermatogenesis from secondary spermatogonia to primary spermatocytes in organ culture of testes fragments from the newt, Cynops pyrrhogaster. The present study demonstrated that FSH promoted in the same model system the differentiation of primary spermatocytes even further: to the stage of elongated spermatids. When testes fragments, consisting of somatic cells and germ cells (mostly primary spermatocytes), were cultured in a control medium for three weeks, only round spermatids and spermatogonia were observed; both the diameter of the cysts and the viability of the germ cells decreased to about 10–15% of the original level. On the other hand, when the medium was supplemented with FSH, elongated spermatids appeared by the second week; both the diameter of the cysts and the viability of the germ cells were maintained at a higher level than in the control medium. The effect of FSH was dose-dependent. However, neither transferrin, androgens (testosterone and 5α-dihydrotestosterone) nor luteinizing hormone (LH) was effective. The addition of cyanoketone, a specific inhibitor of 3β-hydroxy-Δ⁵-steroid dehydrogenase (3β-HSD) (32), to the FSH-containing medium did not prevent the differentiation promoted by FSH, indicating that it is unlikely that Δ⁴-steroid metabolites produced in fragments by FSH acted directly on germ cells. Insulin was found to improve the viability of germ cells during a 2 week of culture period. In the presence of FSH, the cells in various differentiative stages had morphological characteristics very similar to those in vivo, whereas in the absence of FSH primary spermatocytes showed abnormal features in their nuclei and cytoplasm, indicating that they were deteriorating. These results and our previous results (1–3) suggest that FSH promotes primary spermatocytes to differentiate into elongated spermatids probably by stimulating Sertoli cells to secrete factors which then act on the germ cells.     

Abnormalities in the shape of murine sperm after acute testicular x-irradiation

The fraction of murine epididymal sperm with shape abnormalities ranged from 1.0–15.0% in 14 inbred strains and hybrids studied. The effect of x-rays on this fraction was examined in mice with markedly different natural levels of abnormalities, C₅₇BL (9.4%) and (C₅₇BL×C₃H)F₁ (1.0%). Dosages of 30, 100, and 300 rad produced a significant increase in the number of abnormal forms, especially from 3–8 weeks following irradiation. The fraction of abnormal sperm at 5 weeks following graded doses of radiation from 0–300 rad increased from control values to 30%. The data for the hybrid fitted curve which increased as the $\text{sol3}\text{2}$ power of the dose and which gave a doubling in abnormalities compared with control at rad. Sperm abnormalities may permit a simple quantitative assay for damage to the genetic material of the male germ line.     

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.     

Mutagenicity of Ascaris chymotrypsin inhibitor in germ cells of mice

Chymotrypsin inhibitor isolated from Ascaris suum (ACHI) was tested for the induction of dominant lethal mutations in male mice. Dominant lethal effects of ACHI for the main stages of germ cell development were analyzed by mating at specific time points after dosing. Two groups of adult BALB/c males received 24 or 40 mg per kilogram body weight (BW) per day intraperitoneal (IP) injection of ACHI in sterile phosphate-buffered saline (PBS) for five consecutive days (subacute exposure). Males from a third group were administered single IP injections of ACHI—60 mg/kg BW (acute exposure). The control group received concurrent injections of PBS for five successive days. After the last dose, each male was mated with two untreated females. For fractionated examination with regard to successive germ cell stages (spermatozoa, spermatids, spermatocytes, spermatogonia), every second week, two other untreated virgin females were placed with each male for mating. The uteri of the females were inspected on the 15th day of gestation, and preimplantation loss and postimplantation loss determined from dominant lethal parameters. Exposure of mice germ cells to ACHI did not impair mating activity of males. Fertility index was reduced (P < 0.05) only for females mated at the third week with males exposed to the highest dose of ACHI. In the females bred to ACHI-treated males, significant (P < 0.05) increase in preimplantation loss was observed at postinjection weeks 1 (reflecting exposure to spermatozoa after single treatment and to spermatozoa or late spermatids after subacute dosing) and 3 (reflecting exposure to mid and early spermatids for acute dosing and to mid and early spermatids or late spermatocytes following acute treatment), regardless of dose and length of exposure to the inhibitor. At the 60-mg/kg-BW group, a significant increase of this parameter was also noted at week 5 (reflecting exposure to early spermatocytes). During mating days 15–21, a significant (P < 0.05) increase in postimplantation loss and dominant lethal effects were observed for all doses of ACHI. Acute ACHI exposure 5 weeks prior to mating resulted in dominant lethal effects in early spermatocytes. These preliminary data suggest that ACHI induces dominant lethal mutations at postmeiotic and meiotic stages of spermatogenesis, but spermatids are the most sensitive cell stage to the effect of ACHI. These results show that ACHI may be one of the factors causing disturbances in spermatogenesis leading to a reduction of host reproductive success.     

Dose—response relationship for radiation-induced translocations in somatic and germ cells of mice

Dose—response curves (0–600 rad X-rays) for induced reciprocal translocations in bone-marrow cells and in spermatogonia (scored in spermatocytes) of the mouse were constructed. The obtained results suggest that factors influencing aberration yields in somatic cells, are similar to those in germ cells and strengthen the premise for qualitative extrapolation from somatic cells to germ cells.     

A novel germ line-specific gene of the phosducin-like protein (PhLP) family. A meiotic function conserved from yeast to mice

We identified a new member of the phosducin-like (PhLP) protein family that is predominantly, if not exclusively, expressed in male and female germ cells. In situ analysis on testis sections and analysis of purified spermatogenic cell fractions evidenced a stage-specific expression with high levels of RNA and protein in pachytene spermatocytes and round spermatids. Three mRNA species were detected, which correspond to different polyadenylation sites and vary in abundance during germ cell maturation. Only low levels of RNA were detected in whole ovary extracts, but expression of the protein became detectable within hours after hormonal induction of superovulation. The gene (Mgcphlp) is located on mouse chromosome 5 in the immediate vicinity of the Clock locus. The predicted amino acid sequence shows extensive similarities not only with the known mammalian PhLP proteins but also with the yeast phosducin-like protein Plp2, required for the production and growth of haploid cells. Expression of the murine protein was found to complement the defect of a yeast plp2 Delta mutant. We propose that MgcPhLP/Plp2 proteins exert a function in germ cell maturation that is conserved from yeast to mammals.     

Effects of acute and chronic cyclophosphamide treatment on meiotic progression and the induction of DNA double-strand breaks in rat spermatocytes

Male rats treated with cyclophosphamide, an alkylating agent commonly used clinically in both acute and chronic regimens, present with damaged male germ cells and abnormal progeny outcome. The extent and type of damage induced by cyclophosphamide largely depend on the germ cell type exposed to the drug and its ability to respond to insult. In the present study, the response of pachytene spermatocytes to damage was evaluated by assessing their ability to undergo meiotic G2/MI transition following exposure to acute or chronic cyclophosphamide. Male rats were given an acute high dose (70 mg/kg, once) or chronic low doses (6 mg/kg, daily for 5-6 wk) of cyclophosphamide. Pachytene spermatocytes were isolated, cultured, and induced to undergo G2/MI transition with okadaic acid. To determine the effect of DNA damage on meiotic progression, induction of DNA double-strand breaks was detected after each treatment regimen by the formation of foci of phosphorylated histone H2AX. The transition from G2 to MI was impaired after acute cyclophosphamide treatment; this impairment in the progression of pachytene spermatocytes was correlated with extensive DNA double-strand breaks. In contrast, despite the presence of significant levels of DNA damage, meiotic progression was not impaired in spermatocytes after chronic cyclophosphamide exposure. We suggest that the cell cycle impairment induced after acute cyclophosphamide treatment could be mediated by a G2/M checkpoint activated in response to DNA damage. The absence of impairment after chronic treatment raises concern about the functionality of defense mechanisms in male germ cells after repeated exposure to low doses of genotoxic agents.