Mutagenesis, Vol. 17, No. 1, 9-13,
January 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
Germline drift in chimeric male mice possessing an F2 component with a paternal F0 radiation history
Institute of Toxicology and Environmental Health, University of California, Davis, CA 95616, USA
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Abstract |
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In earlier work using mouse chimeric embryos we have shown that pre-implantation embryonic cells with a history of paternal
-irradiation (1 Gy) 6 weeks prior to conception demonstrated a competitive cell proliferation disadvantage when challenged by direct cell–cell contact with control pre-implantation embryos in chimeras even two generations after paternal irradiation. This effect on embryonic cell proliferation was associated with paternal irradiation of sensitive type A/B spermatogonia 6 weeks prior to conception but not from irradiation 5 weeks prior to conception. The purpose of this new study is to test the hypothesis that germ cell lines with a history of acute irradiation may also exhibit a selection disadvantage or germline chimeric drift over time in adult male mice two generations after paternal irradiation in chimeric mice. F0 male CD1 mice received a 1.0 Gy whole body absorbed dose of attenuated 137Cs -rays and were mated at post-irradiation weeks 5 and 6. Chimeric XY
XY male CD1 mice were constructed with F2 embryos from the F1 offspring and with control embryos. To distinguish the control germline cells, the control embryos were heterozygous for the neo transgene, which served as a cell lineage marker. To test for germline chimeric drift, each XYXY chimera was mated periodically from age 7 to 21 weeks with a different CD1 female. The fraction of offspring from each liter with the neo marker was used to quantify the relative contribution of the control germ cell lineage to the fertilizing sperm population. The results showed that there was a significant selection against germ cells with the paternal F0 post-irradiation week 6 history. In contrast, there was a very small but significant selection in favor of the germ cells with the paternal F0 post-irradiation week 5 history. These results indicate that the effects of a relatively non-toxic dose of radiation (1 Gy) on cell proliferation transmitted by ancestral type B spermatogonia to the embryo are manifested in the germline of the adult male mice even after two generations. |
Introduction |
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Long-standing concern over the potential for genetic effects from exposure to ionizing radiation has led to extensive study of germline mutations (United Nations Scientific Committee on the Effects of Atomic Radiation, 1986
, 2000). In previous studies the pre-implantation embryo chimera assay has been used to demonstrate effects on pre-implantation embryos that were sired by males exposed to ionizing radiation during radiation-sensitive stages of spermatogenesis, associated with type A/B spermatogonia and pachytene spermatocytes. That assay utilized aggregation chimeras constructed from a 4 cell embryo with a radiation history together with a control 4 cell embryo. The assay measures the competitive cell proliferation disadvantage of an experimental embryo when challenged by direct cell–cell contact with a normal embryo (Obasaju et al., 1988, 1989; Warner et al., 1991; Straume et al., 1993, 1997; Wiley et al., 1994a,b, 1997; Peters et al., 1996). A competitive cell proliferation disadvantage of progeny embryos has been demonstrated following acute paternal whole body exposure to low LET radiation [X-rays (Obasaju et al., 1989) and -rays (Warner et al., 1991)] or high LET radiation [512 MeV/a.m.u. 56Fe (Wiley et al., 1994a)] with absorbed doses of 0.01–1.0 Gy. This large body of work demonstrates that the sensitivity of the pre-implantation embryo chimera assay in detecting the transmission of biological effects of both low and high doses of ionizing radiation on sensitive spermatogenic stages is surprisingly high, at ~0.3. Statistically significant effects have repeatedly been detected using 5–10 animals/dose group and 5–10 chimeras/animal. In contrast, the mouse specific locus assay required thousands of animals in order to detect heritable mutations with a transmission frequency of ~10-5 following doses of ~3 Gy (Russell and Kelly, 1982).
The pre-implantation embryo chimera assay has been used to demonstrate that following paternal irradiation yielding doses of from 0.01 to 0.05 Gy the embryos conceived 4 and 6–7 weeks later have a significant competitive cell proliferation disadvantage (Warner et al., 1991). This temporal pattern suggests that the embryonic effect is transmitted by sperm that were pachytene spermatocytes and late type A/early type B spermatogonia, respectively, at the time of irradiation (Obasaju et al., 1989). These same stages of spermatogenesis have been implicated in the transmission of genetic effects and are also the most susceptible to cell killing following paternal irradiation (Matsuda et al., 1985; Nomura, 1988; Meistrich and Van Beek, 1990; Burruel et al., 1997). The cell proliferation disadvantage that is transmitted to the F1 embryo following paternal F0 irradiation also appears to be inherited by the F2 embryo with unexpectedly high frequencies of occurrence as compared with transmission frequencies that would be associated with radiation-induced mutations at specific loci (Wiley et al., 1997). This lack of degradation in transmission frequency of pre-implantation embryo competitive cell proliferation disadvantage between the F1 and F2 generations suggests a non-Mendelian mode of inheritance or genomic instability initiated by irradiation of the paternal F0 germline.
We hypothesized that the biological mechanisms responsible for the competitive cell proliferation disadvantage of an F2 embryo with a paternal F0 post-irradiation week 6 radiation history were not just specific to the embryo and that the effects of paternal F0 irradiation would persist beyond embryonic development in the chimeric offspring. Hence, our test hypothesis was that the cell proliferation disadvantage that has been observed in both F1 and F2 embryo studies would also be manifested in adult chimeric animals. To test this hypothesis, we constructed aggregation chimeras composed of F2 embryos with a radiation history and control embryos with no radiation history. The control embryos with no radiation history carried a transgene that enabled us to identify their cell lines in the chimeric offspring. After transfer of the chimeric embryos to foster mothers, the resulting male chimeric offspring were studied for evidence of chimeric drift in the germline. The results of this pilot study suggest that the germ cell lines with a history of acute radiation exposure of type B spermatogonia were selected against over time in adult F2 chimeric mice.
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Materials and methods |
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The materials and methods regarding the mice, irradiations and post-irradiation breedings to produce F1 males and their F2 embryos were as described in Baulch et al. (2001) except that in this study the mated females from both 5 and 6 weeks after paternal F0 irradiation were allowed to deliver their F1 litters. Post-irradiation week 5 was considered the control mating for this experiment, since pre-implantation embryo chimera assays using 1.0 Gy of 137Cs
-radiation demonstrated that F1 and F2 paternal F0 post-irradiation week 5 embryos exhibit no competitive cell proliferation disadvantage (Wiley et al., 1997). Additionally, comparison of two different spermatogenic stages from the same sires reduces extraneous biological variability and emphasizes the difference in effect on heritable competitive cell proliferation disadvantage of the two spermatogenic stages based upon the sensitivity of each spermatogenic stage to acute -irradiation.
Production of chimeric embryos and offspring
Chimeric embryos were constructed and transferred to foster mothers in order to evaluate `chimeric drift' in the germline of the male offspring. Chimeras were constructed from one F2 embryo with a paternal F0 radiation history and one control embryo with no paternal radiation history. Some cell lines from the control embryo could be subsequently identified because the control embryo was heterozygous for the neo transgene (neo+/–) (Figure 1). Offspring resulting from the transfer of chimeric embryos to recipient females were screened by PCR to determine the sex of each animal (possible outcomes were XXXX, XXXY and XYXY) (Aasen and Medrano, 1990; Peippo and Bredbacka, 1995). The XYXY chimeric offspring that tested positive for the neo cell lineage marker in tail DNA were each bred periodically from 7 to 21 weeks of age to a different superovulated CD1 female. All offspring from each litter were screened for the neo marker. The change in the proportion of neo+ offspring per litter over time was used as a measure of germ cell chimeric drift in the sperm population of the chimeric F2 sire as he aged. The data were recorded and evaluated separately for each sire, then jointly analyzed by experimental group.
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Testing of transgenic animals
Based on the observations described in Baulch et al. (2001), the neo transgene was used in the heterozygous state. Because the neo+/– control embryo used to construct the chimeras was heterozygous for the neo marker, only half of the offspring derived from the control cell lineage carried the neo marker and could be identified in this study. The remaining neo-/- control offspring were indistinguishable from the neo-/- F3 offspring with a paternal F0 radiation history. A significant increase in the proportion of the neo+ offspring over time was considered evidence of chimeric drift with selection against the germline having a radiation history.
DNA purification and PCR for the neo marker
DNA was purified from mouse tail clips using the basic QiaAmp Tissue Kit and protocol (Qiagen, Valencia, CA; catalog no. 29306).
Using the 21 nt sense primer 5'-CGCCTTCTATCGCCTTCTTGA-3' and the 21 nt antisense primer 5'-CTCCCCACCCGTTTTCCTCTG-3' (GENSET, La Jolla, CA) a 357 bp neo PCR product was obtained and verified by BamHI restriction enzyme digestion (Promega, Madison, WI).
Statistical analyses
The chimeric drift statistical analysis involves Student's t-test comparisons of the observed slopes of the linearized fractions of germline types as a function of time for the two paternal F0 -irradiation times (post-irradiation weeks 5 and 6). The slopes and their variances were obtained by weighted least squares regression analysis with the data points appropriately weighted with respect to litter size and statistical uncertainty. The observed neo+ fraction for each sire for each week was calculated as a binomial distribution and the total number of offspring in the litters was used to calculate the variance of the observed neo+ ratios using binomial distribution analysis. This variance was used to properly weight the datum entered in the weighted least squares regression analysis.
A preliminary weighted least squares linear regression of the observed data for each chimeric sire as a function of paternal age was performed before evaluating the germline chimeric drift data. A scaling correction was added to normalize the y-intercept (extrapolated neo+ fraction at paternal age 0) of each regression to 0.5 for the nominal neo+ litter fraction without altering the regression slope. The normalized data from each group (paternal F0 post-irradiation weeks 5 and 6) were separately analyzed as a function of paternal age by linear regression, using weighted least squares, where each weight was the inverse of the estimated variance of the values obtained from the observed proportion neo+ for each litter. The resulting slopes of the two fitted lines were contrasted with each other by Student's t-test. In addition, the slope of each line was compared to slope = 0 (zero correlation or no drift) by Student's t-test. Error bars represent the standard error of all data for a given paternal age, where data represent more than one litter, or the standard deviation based on the neo binomial ratio where only one litter was represented. All statistical tests utilized P = 0.05 significance levels.
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Results |
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Out of 30 chimeric offspring, a total of 10 XY
XY chimeric males were obtained (Table I). One male from each experimental group showed no evidence of neo+ sperm. One male from the group with a paternal F0 post-irradiation week 6 history showed no evidence of neo- sperm from a radiation history as demonstrated by peak elevation of neo+ ratios at all four time points measured (0.44–0.64, Table II). These three animals were excluded from the final statistical analyses. As a result, the data used to evaluate germline chimeric drift were obtained from three males derived from F2 embryos with three different F1 sires having a paternal F0 post-irradiation week 5 history and four males derived from F2 embryos with three different F1 sires having a paternal F0 post-irradiation week 6 history. These seven males that had a paternal F0 post-irradiation week 5 or 6 history were mated six times over 13 weeks to obtain a minimum of three litters per chimeric sire (Table II).
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The weighted least squares regression analysis of the proportions of the litters for each of the two experimental groups yielded slopes of –0.010 ± 0.004 (SE) and 0.064 ± 0.011 (SE) for a F0 post-irradiation week 5 history and a F0 post-irradiation week 6 history, respectively (Figure 2
). The paternal F0 post-irradiation week 6 history group had a significantly steeper slope in comparison with the paternal F0 post-irradiation week 5 history group (P << 0.001, one-tailed Student's t-test). When compared with the zero slope (no drift), the positive slope of the line for the week 6 group and the small negative slope for the week 5 group were both significant (P < 0.001, one-tailed Student's t-test, and P< 0.1, two-tailed Student's t-test, respectively). While the changes in the neo+ fraction of both groups were significantly correlated with F2 paternal age, the week 6 group showed selection against the irradiated component and the week 5 group showed slight, but significant, selection against the neo component.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Fewer neo- offspring were produced over time by the chimeric males with a germline component descended from offspring conceived at paternal F0 post-irradiation week 6, corresponding to irradiation of the sensitive paternal F0 type B spermatogonia. In contrast, there was little overall change in the number of neo- offspring produced by the males with a germline component descended from paternal F0 post-irradiation week 5, corresponding to irradiation of the relatively radiation-resistant early spermatocytes. This observation is consistent with results of the pre-implantation embryo chimera assay, which also revealed little or no effect on the cell proliferation of F1 or F2 embryos produced by sperm at paternal F0 post-irradiation week 5 (Wiley et al., 1997
). The decreasing relative numbers of neo- offspring that were observed in the litters from the week 6 chimeric males are unlikely to have resulted from prenatal death since there were no significant changes in litter size over the 13 weeks of the experiment (Table II
). When the proportion of neo+ offspring in the litters was evaluated versus time, the chimeric males with a germline component descended from paternal F0 post-irradiation week 6 had a line with positive slope that was significantly different from 0. A line with a slope significantly different from 0 is evidence for chimeric drift and, in this study, a line with a positive slope is consistent with a competitive cell proliferation disadvantage of the germ cells with a paternal F0 radiation history.
The simplest interpretation of these results is that chimeric drift has occurred against the neo- germ cell population inheriting the paternal F0 post-irradiation week 6 radiation exposure (Figure 2). It is unlikely that these results are due to a selection for the neo marker, since the slight chimeric drift observed in the F0 post-irradiation week 5 history group was in the negative direction. The latter result suggests either a selection against the neo marker or that there is a selective advantage to some stages of spermatogenesis of inheriting a history of exposure to certain doses of radiation. Factors other than the inherited effect of paternal F0 irradiation that might influence germ cell proliferation rates would have affected both the neo+ germ cells and the neo- germ cells with the radiation history equally, since both spermatogenic cell types resided within the same gonad.
Also, the results of this study may be more meaningful than our data demonstrate because our inability to distinguish the neo-/- F3 offspring with a paternal F0 radiation history from the neo-/- control offspring reduces the statistical power of the analysis.
These results are consistent with the hypothesis that the selection against germ cells with the paternal F0 post-irradiation week 6 history represents the radiation response of the ancestral type B spermatogonia, the spermatogonial stage that also transmits the greatest degree of competitive cell proliferation disadvantage to F1 and F2 embryos in pre-implantation embryo chimera assays (Obasaju et al., 1989; Warner et al., 1991; Wiley et al., 1997). Conversely, the paternal F0 post-irradiation week 5 history, which is not associated with any significant competitive cell proliferation disadvantage in F1 and F2 embryos, is not associated with chimeric drift against neo- germ cells in chimeric males. This is why post-irradiation week 5 has generally been used as an internal control for embryonic cell proliferation disadvantage in chimera assays (Wiley et al., 1997).
Genomic instability may be the mechanism underlying the chimeric drift, or selection against germ cells with the paternal F0 post-irradiation week 6 history, observed in these germ cells as a result of their paternal F0 radiation history. If irradiation of the paternal F0 type B spermatogonia initiated genomic instability, then the germ cells with a paternal F0 post-irradiation week 6 history may be less fit. Over time, the genetically unstable germ cells might lose the cellular proliferation competition to the genetically stable germ cells from the control component of the chimeric gonad (Carls and Schiestl, 1999).
The results reported here also suggest that the pre-implantation embryo chimera assay may be predictive of heritable cellular effects from irradiation of the ancestral type B spermatogonia, not just in the pre-implantation embryo, but in live born offspring. In fact, in another study we have recently demonstrated that 19-day-old F3 offspring from chimeric F2 sires with a component from a paternal F0 post-irradiation week 6 history had significant alterations in the hepatic activities of receptor tyrosine kinase, protein kinase C and mitogen-activated protein kinase, in comparison with neo+ littermate controls (Baulch et al., 2001). These offspring also had significantly elevated nuclear protein levels of p53 and p21waf1.
Our data and the data from other laboratories support the hypothesis that paternal germline irradiation induces genomic instability in offspring (Carls and Schiestl, 1999; Dubrova et al., 2000; Baulch et al., 2001). Future studies with much larger numbers of animals will be needed to confirm the heritability of a competitive cell proliferation disadvantage in germ cells following paternal F0 irradiation and to elucidate the underlying mechanism. Since both embryonic and spermatogenic cells are extremely specialized cell types, it is important that a somatic cell population, such as nucleated cells in peripheral blood, from chimeric animals also be evaluated for chimeric drift in these studies.
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Acknowledgments |
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The authors wish to acknowledge the expert technical assistance of Marie Suffia and George Withers in the production of the chimeric F2 XY–XY male mice. This work was supported by PHS grant R01ES06516 to L.M.W. and J.W.O. and NIEHS grant 5 T32 ES07059 to J.E.B.
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Notes |
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1 To whom correspondence should be addressed. Tel: +1 530 752 9872; Fax: +1 530 752 5300; E-mail: jebaulch{at}uedavis.edu
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References |
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Received on April 9, 2001; accepted on August 8, 2001.
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