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.     

The induction of specific-locus mutations with N-propyl-N-nitrosourea in stem-cell spermatogonia of mice.

A specific-locus test to determine the effect of N-propyl-N-nitrosourea (PNU) on the stem-cell spermatogonia of mice has been performed. Male wild-type mice (C3H/He) were treated with an intraperitoneal injection of 200 mg/kg [corrected] of PNU. Eight weeks after the injections, the males were mated with tester stock females (PW), homozygous for 6 visible recessive genes. Twelve mutants among 8605 offspring were observed. The mutation frequency with PNU was calculated to be 23.2 x 10(-5)/locus/gamete, showing a significant difference from that of the non-treated control. The mutations were all heritable and half of them were viable in homozygous condition. The mutation frequency with PNU was about one-third of that with N-ethyl-N-nitrosourea, a highly potent mutagen for mouse stem-cell spermatogonia.     

Comparison of two stocks of mice in spermatogonial response to different conditions of radiation exposure

In a previous report (Generoso et al., 1985) it was shown that the two hybrid stocks of mice, (C3H/R1 x 101/R1)F1 and (SEC/R1 x C57BL/6)F1, differed in their responses to induction of chromosomal aberrations following exposure of the stem-cell spermatogonia to 500 R x 4 (4-week intervals) acute X-rays. The levels of response in the two stocks were paralleled by the effects on the length of the sterile period, which presumably results from stem-cell killing and repopulation. The present study was conducted in order to determine whether the differences between the two stocks in these parameters hold true also for other conditions of radiation exposure. Thus, comparative experiments were conducted using the following acute exposure regimens: 500 R single dose, 500 R + 500 R (24-h interval), 100 R + 900 R (24-h interval), and 500 R x 4 (8-week intervals). The endpoints measured were chromosome rearrangements in diakinesis/metaphase-I meiocytes, embryonic lethality in conceptuses, length of sterile period and testis weight. Trend analysis indicated that higher frequencies of chromosome rearrangements and embryonic lethality were recovered from (C3H/R1 x 101/R1)F1 than from (SEC/R1 x C57BL/6)F1 males, that there were no significant differences between stocks in testis weight reductions, and that there was no consistency in the direction of the significant differences that occurred in the length of the sterile period. A definitive conclusion regarding the possible association between induction of chromosomal aberrations and induction of cell killing awaits direct histological analysis of the stem-cell population.     

Absence of correlation between the chromosomal radiosensitivity of peripheral blood lymphocytes and stem-cell spermatogonia in mammals

To evaluate the reliability of quantitative extrapolation of radiation-induced chromosomal damage from somatic cells to germ cells, data on the effects of several biological and physical factors on the chromosomal radiosensitivity of blood lymphocytes and stem-cell spermatogonia have been collected from the literature. The results show that most of the factors considered, such as chromosomal constitution, age, genetic constitution, species, sampling time and dose fractionation, had differential effects on the induction of chromosomal aberrations in both systems. These differential effects can easily be explained in terms of the biological differences between in-vitro-stimulated peripheral blood lymphocytes and stem-cell spermatogonia. It is concluded that only direct experiments on germ cells of higher primates and man can be used for a quantitative estimation of human genetic radiation risks arising from structural chromosomal aberrations.     

Effect of X-ray and ethylnitrosourea exposures separated by 24 h on specific-locus mutation frequency in mouse stem-cell spermatogonia

Specific-locus mutation frequencies in mouse stem-cell spermatogonia were determined in 3 experiments in which mature male mice were exposed to 100,m 300, or 500 R of X-rays followed, 24 h later, by intraperitoneal injection of 100 mg/kg of ethylnitrosourea (ENU). The purpose was to find out if the mutation frequencies would be augmented over those expected on the basis of additivity of the effects of the separate treatments. Such augmentation had been observed in earlier work in which exposure to 100 or 500 R of X-rays was followed 24 h later by a second exposure of 500 R. No augmentation was observed for X-rays followed by ENU. The mutation frequencies in all 3 experiments actually fell below those expected on the basis of additivity, although the reductions were not statistically significant.     

Clonal analysis of radiation-induced translocations in stem-cell spermatogonia of normal and T70H translocation heterozygous mice

7 T(1;13)70H/+ and 13 +/+ male mice were given 2 doses of 250 rad acute X-rays separated by 24 h. The +/+ mice were analysed in 2 groups during the first meiotic division for induced translocations, on average 177 and 233 days after irradiation, and the T70H/+ mice were analysed in parallel with the second group of +/+ males. One testis was treated with normal air-drying procedures yielding a random sample of cells. The other testis was processed according to a new technique, which enabled separate analysis of the various locations along the seminiferous epithelium where groups of cells are synchronously in the diakinesis-metaphase I stage of meiosis. The number of cells in such groups was estimated. Both capita epididymes were used for a sperm count. In agreement with an earlier finding, fewer induced translocations were recovered from the T70H/+ mice than from +/+ mice (10.6 versus 19.2%, air-drying technique).Estimates of the group sizes in combination with the occurrence of induced translocations yielded the following information. A synchronously moving group of diakinesis-metaphase I cells originates from, on average, 1.25 stem cells (Appendix). We found an indication for a reduction in group size by 33% when a clone originated from a stem cell carrying an induced translocation compared with a wild-type clone (see Appendix). Both, the data on group size and the sperm counts indicate that, 7 months after the irradiation, the seminiferous epithelium has not totally recovered. Final recovery seems to be slower or absent in the T70H/+ males. The data obtained from the T70H/+ heterozygotes indicate the stem-cell spermatogonia to be responsible for the reduction of the rate of translocation induction with this karyotype, either due to a reduced formation rate or due to a diminished capacity of some of the induced translocation-carrying stem cells to proliferate into a clone reaching the meiotic divisions.     

Interactions between phencyclidine and delta 9-tetrahydrocannabinol in mice following smoke exposure

The behavioral and pharmacological interactions between delta 9-tetrahydrocannabinol (delta 9-THC) and phencyclidine (PCP) were studied following coadministration of the drugs in smoke to mice. While delta 9-THC (25, 50 or 100 mg/cigarette) had little effect on spontaneous motor activity, all doses attenuated the hyperactivity elicited by PCP X HCl (25 and 50 mg/cigarette). delta 9-THC produced a dose-related hypothermia. PCP X HCl (50 mg/cigarette) had no effect on body temperature but enhanced hypothermia when combined with 25 mg of delta 9-THC. delta 9-THC (100 mg/cigarette) had no effect on the biodisposition of 3H-PCP and its pyrolytic product, 3H-phenylcyclohexene (3H-PC), when examined immediately after 3H-PCP X HCl (50 mg/ cigarette) exposure. At 30 min, brain, liver, lung and plasma contained higher concentrations of 3H-PC and fat and plasma contained lower concentrations of 3H-PCP in the mice exposed to both drugs compared to 3H-PCP X HCl alone. It appears, therefore, that delta 9-THC has the potential for altering the behavioral, pharmacological and pharmacokinetic sequelae of PCP abuse.     

Estimation of coefficients in a model of radiation-induced myelopoiesis from mortality data for mice following X-ray exposure

The rate coefficients in the model of cell kinetics and mortality introduced by Jones et al. (Radiat. Res. 128, 258-266 (1991)) are estimated using mortality data from several mouse experiments. The evaluated model fits data from a large variety of prompt, protracted, and fractionated irradiations with 250-k Vp X rays with good fidelity. Although the maximum-likelihood estimates are not unique, all estimates lead to greater cell survival than that observed in in vitro experiments on nonterminally differentiated reproducing cells from the marrow.     

Maternal nicotine exposure induces congenital heart defects in the offspring of mice

Maternal cigarette smoking is a risk factor for congenital heart defects (CHDs). Nicotine replacement therapies are often offered to pregnant women following failed attempts of smoking cessation. However, the impact of nicotine on embryonic heart development is not well understood. In the present study, the effects of maternal nicotine exposure (MNE) during pregnancy on foetal heart morphogenesis were studied. Adult female mice were treated with nicotine using subcutaneous osmotic pumps at 0.75 or 1.5 mg/kg/day and subsequently bred with male mice. Our results show that MNE dose‐dependently increased CHDs in foetal mice. CHDs included atrial and ventricular septal defects, double outlet right ventricle, unguarded tricuspid orifice, hypoplastic left ventricle, thickened aortic and pulmonary valves, and ventricular hypertrophy. MNE also significantly reduced coronary artery size and vessel abundance in foetal hearts. Moreover, MNE resulted in higher levels of oxidative stress and altered the expression of key cardiogenic regulators in the developing heart. Nicotine exposure reduced epicardial‐to‐mesenchymal transition in foetal hearts. In conclusion, MNE induces CHDs and coronary artery malformation in mice. These findings provide insight into the adverse outcomes of foetuses by MNE during pregnancy.     

Rescue of developmental lens abnormalities in chimaeras of noncataractous and congenital cataractous mice

In the study of the lens of a congenital cataractous mouse mutant (CAT), it has been shown that a loss of growth regulation at the cellular level causes gross lens abnormalities. The phenotypic characteristics of the cataractous mouse lens are similar to those seen in human congenital cataract and thus serves as a model system for medical research. In this present investigation, we have demonstrated that the abnormalities of the congenital cataractous lens can be rescued by forming chimaeras between DBA/2 (a noncataractous strain of mouse) and the CAT mutant. This report describes the histological, cellular and biochemical analysis of the resultant chimaeric eyes, and discusses possible mechanisms by which these results were achieved.     

Differential patterns of introgression across the X chromosome in a hybrid zone between two species of house mice

A complete understanding of the speciation process requires the identification of genomic regions and genes that confer reproductive barriers between species. Empirical and theoretical research has revealed two important patterns in the evolution of reproductive isolation in animals: isolation typically arises as a result of disrupted epistatic interactions between multiple loci and these disruptions map disproportionately to the X chromosome. These patterns suggest that a targeted examination of natural gene flow between closely related species at X-linked markers with known positions would provide insight into the genetic basis of speciation. We take advantage of the existence of genomic data and a well-documented European zone of hybridization between two species of house mice, Mus domesticus and M. musculus, to conduct such a survey. We evaluate patterns of introgression across the hybrid zone for 13 diagnostic X-linked loci with known chromosomal positions using a maximum likelihood model. Interlocus comparisons clearly identify one locus with reduced introgression across the center of the hybrid zone, pinpointing a candidate region for reproductive isolation. Results also reveal one locus with high frequencies of M. domesticus alleles in populations on the M. musculus side of the zone, suggesting the possibility that positive selection may act to drive the spread of alleles from one species on to the genomic background of the other species. Finally, cline width and cline center are strongly positively correlated across the X chromosome, indicating that gene flow of the X chromosome may be asymmetrical. This study highlights the utility of natural populations of hybrids for mapping speciation genes and suggests that the middle of the X chromosome may be important for reproductive isolation between species of house mice.     

Tissue-specific variation in repair activity for 3-methyladenine in DNA in two stocks of mice

Two stocks of mice, hybrid (C3H X 101)F1 and inbred SEC/R1, were compared for 3-methyladenine-DNA N-glycosylase activity which is involved in removal of 3-methyladenine, 7-methylguanine and some other N-methylpurines in DNA, in cell-free extracts of different tissues. Based on activity measured both per unit weight of tissue and per mass DNA, there is a significant organ-specific and stock-specific difference in N-glycosylase activity over a range of 0.5-8.7 fmoles of 3-methyladenine released per h at 37 degrees C per micrograms DNA of tissue extract. On a per cell basis, the repair activity for 3-methyladenine is the highest in stomach in both stocks. The tissue can be arranged in order of decreasing activity of glycolytic removal as stomach greater than kidney greater than lung greater than liver greater than spleen greater than brain greater than ovary for SEC/R1 mice and stomach greater than kidney greater than ovary greater than spleen, lung and brain greater than liver for the hybrid mice. For all tissues except ovary, SEC/R1 mice have 1.5-4-fold higher specific N-glycosylase activity than (C3H X 101)F1 mice. In contrast, the ovary of SEC/R1 stock has about half as much enzyme activity as that of the hybrid stock.     

A complex genetic basis to X-linked hybrid male sterility between two species of house mice

The X chromosome plays a central role in the evolution of reproductive isolation, but few studies have examined the genetic basis of X-linked incompatibilities during the early stages of speciation. We report the results of a large experiment focused on the reciprocal introgression of the X chromosome between two species of house mice, Mus musculus and M. domesticus. Introgression of the M. musculus X chromosome into a wild-derived M. domesticus genetic background produced male-limited sterility, qualitatively consistent with previous experiments using classic inbred strains to represent M. domesticus. The genetic basis of sterility involved a minimum of four X-linked factors. The phenotypic effects of major sterility QTL were largely additive and resulted in complete sterility when combined. No sterility factors were uncovered on the M. domesticus X chromosome. Overall, these results revealed a complex and asymmetric genetic basis to X-linked hybrid male sterility during the early stages of speciation in mice. Combined with data from previous studies, we identify one relatively narrow interval on the M. musculus X chromosome involved in hybrid male sterility. Only a handful of spermatogenic genes are within this region, including one of the most rapidly evolving genes on the mouse X chromosome.