Risk estimation based on germ-cell mutations in animals

The set of mouse germ cell mutation rate results following spermatogonial exposure to high dose rate irradiation have been presented as the most relevant experimental results upon which to extrapolate the expected genetic risk of offspring of the survivors of the Hiroshima and Nagasaki atomic bombings. Results include mutation rates to recessive specific-locus, dominant cataract, protein-charge, and enzyme-activity alleles. The mutability as determined by the various genetic end points differed: the mutation rates to recessive specific-locus alleles and enzyme-activity alleles were similar and greater than the mutation rates to dominant cataract and protein-charge alleles. It is argued that the type of mutation event scored by a particular test will determine the mutability of the genetic end point screened. When the loss of functional gene product can be scored in a particular mutation test, as in the recessive specific-locus and enzyme-activity tests, a wide spectrum of DNA alterations may result in a loss of and a higher mutation rate is observed. When an altered gene product is scored, as in the dominant cataract and protein-charge tests, a narrower spectrum of DNA alterations is screened and a lower mutation rate is observed. The radiation doubling dose, defined as the dose that induces as many mutations as occur spontaneously per generation, was shown to be four times higher in the dominant cataract test than the specific-locus test. These results indicate that to extrapolate to genetic risks in humans using the doubling-dose method, the extrapolation must be based on experimental mutation rate results for the same genetic end point.(ABSTRACT TRUNCATED AT 250 WORDS)     

Linear dose-response relationship of erythrocyte enzyme-activity mutations in offspring of ethylnitrosourea-treated mice

The specific activity of 10 erythrocyte enzymes was measured to detect gene mutations in F1 offspring of male mice treated with 3 different doses of ethylnitrosourea (ENU). After administration of ENU or of the solvent (controls), the (101/El X C3H/El)F1 hybrid males were mated to untreated T-stock females. No enzyme-activity mutant was found in 3610 F1 offspring of the control group. After treatment of postspermatogonial germ-cell stages, 1 mutant in 1125 F1 offspring of males treated with 160 mg ENU/kg body weight, and 2 mutants in 1319 F1 offspring of a 250-mg/kg group were observed. After treatment of spermatogonia, 9 enzyme-activity mutants in 4247 F1 offspring of males treated with 80 mg ENU/kg body weight, 15 mutants in 3396 F1 offspring of a 160-mg/kg group, and 9 mutants in 1402 F1 offspring of a 250-mg/kg group were detected. The mutation frequencies in spermatogonia were significantly different from that of the controls (P less than 0.01). The dose-response curve was found to be linear. The frequencies of enzyme-activity mutations are comparable to those of recessive specific-locus mutations determined in the same experiments. Enzyme-activity mutants with reduced activity as well as mutants with enhanced activity were found. Genetic and biochemical characterization of enzyme-activity mutants was routinely performed. In inter se crossings of heterozygotes, no offspring expressing a third phenotype other than the wild type and the heterozygote were found in approximately half of the mutation studies. The recovered mouse mutants might be used as animal models to study corresponding genetic diseases in humans.     

The paternal genome in mouse zygotes is less sensitive to ENU mutagenesis than the maternal genome

The two parental genomes lie separate within the zygote and may be differentially affected by environmental influences. We have shown earlier (Russell et al., 1988) that the maternal genome within the mouse zygote is exquisitely sensitive to the induction of point mutations by N-ethyl-N-nitrosourea (ENU), and that the initial lesion probably occurs in one strand of the DNA. The present experiment measured specific-locus mutation induction in the paternal genome. Zygotes containing a multiple-recessive maternal genome (a; b; p cch; d se; s) and the corresponding wild-type alleles in the paternal one were exposed to 50 mg ENU/kg in vivo at one of two stages: the presumed times of sperm entry and early pronuclear stage. At weaning age, the resulting mice were examined for mutations at the marked loci as well as for other mutations producing externally visible phenotypes. At the marked loci, one possible mosaic (for b) was observed among 2113 classified offspring that had been treated with ENU as zygotes; this animal failed to transmit a mutation. By contrast, in the reciprocal cross (which tests the maternal genome) we had observed 8 specific-locus mutations (6 of them mosaics) among 1555 offspring that had received the same dose of ENU during sperm entry (and completion of oocyte meiosis II). In the present experiment, we also found one mutation at other loci (two at other loci in the reciprocal cross). The frequency of offspring with small white belly spots was significantly greater in the treated groups (3.5 and 1.9% at the earlier and later stage, respectively) than in the control (1.0%), the excess being almost entirely due to daughters. Genetic tests of a large number of such offspring failed to find a genetic cause. Instead, it appears that this phenotype may be influenced by factors in the intrauterine environment. It is concluded that shortly after sperm entry, the paternal genome of the zygote is less sensitive than the maternal one to the induction of mutations by ENU.     

Towards an understanding of the nature and fitness of induced mutations in germ cells of mice: homozygous viability and heterozygous fitness effects of induced specific-locus, dominant cataract and enzyme-activity mutations

A total of 219 specific-locus, 35 dominant cataract and 44 enzyme-activity mutations induced in spermatogonia of mice by radiation or ethylnitrosourea (ENU) treatment were characterized for homozygous viability as well as fitness effects on heterozygous carriers. For all 3 genetic endpoints, the frequency of homozygous lethal mutations was higher in the group of radiation-induced mutations than in the ENU-treatment group. These observations are consistent with the hypothesis that radiation-induced mutations recovered in the mouse are mainly due to small deletions while ENU induces mainly intragenic mutations. The overall fitness of mutant heterozygotes was reduced for the group of radiation-induced specific-locus, dominant cataract and enzyme-activity mutations while the ENU-induced mutations exhibited no reduction in fitness. The fitness reduction of heterozygous carriers for a newly occurring mutation in a population is important in determining the persistence of the mutation in a population, and thus the total number of individuals affected before a mutation is eventually eliminated from the population. For the present results a maximal persistence of 12 generations and a minimal persistence of 3 generations is estimated. These results are consistent with the 6-7-generation persistence time assumed by UNSCEAR (1982) in an estimate of the overall effects of radiation-induced mutations in man.     

Efficient Recovery of Enu-Induced Mutations from the Zebrafish Germline

We studied the efficiency with which two chemical mutagens, ethyl methanesulfonate (EMS) and N-ethyl-N-nitrosourea (ENU) can induce mutations at different stages of spermatogenesis in zebrafish (Brachydanio rerio). Both EMS and ENU induced mutations at high rates in post-meiotic germ cells, as indicated by the incidence of F(1) progeny mosaic for the albino mutation. For pre-meiotic germ cells, however, only ENU was found to be an effective mutagen, as indicated by the frequencies of non-mosaic mutant progeny at four different pigmentation loci. Several mutagenic regimens that varied in either the number of treatments or the concentration of ENU were studied to achieve an optimal ratio between the mutagenicity and toxicity. For the two most mutagenic regimens: 4 X 1 hr in 3 mM ENU and 6 X 1 hr in 3 mM ENU, the minimum estimate of frequencies of independent mutations per locus per gamete was 0.9-1.3 X 10(-3). We demonstrate that embryonic lethal mutations induced with ENU were transmitted to offspring and that they could be recovered in an F(2) screen. An average frequency of specific-locus mutations of 1.1 X 10(-3) corresponded to approximately 1.7 embryonic lethal mutations per single mutagenized genome. The high rates of mutations achievable with ENU allow for rapid identification of large numbers of genes involved in a variety of aspects of zebrafish development.     

The induction of forward and reverse specific-locus mutations and dominant cataract mutations in spermatogonia of treated strain DBA/2 mice by ethylnitrosourea

The mutagenic effectiveness of ethylnitrosurea (ENU) was assessed in treated spermatogonia of DBA/2 mice. In a total of 17,515 offspring examined following 160 mg ENU/kg body weight treatment of parental males, 26 forward specific-locus mutations, 2 reverse specific-locus mutations and 9 dominant cataract mutations were recovered. ENU increased the mutation rate to all 3 genetic endpoints. However, ENU was less effective in treated DBA/2 mice than in the standard experimental protocol employing treated hybrid (102 X C3H)F1 male mice. This observed difference for a direct-acting mutagen such as ENU may result from differences in the detoxification of ENU or from differences in the DNA-repair capabilities of strain DBA/2. The first documented reverse mutation of the b allele is reported. The reversion was shown to be due to an AT to GC transition. To date, in addition to the reverse mutation of the b allele, 5 independent ENU-induced mutations recovered in germ cells of the mouse have been molecularly characterized and all have been shown to be base substitutions at an AT site. This is in contrast to the expected mechanism of ENU mutation induction due to O6-ethylguanine adduct formation which results in a GC to AT base-pair substitution and emphasizes the complexities of mutagenesis in germ cells of mammals.     

Induction of specific-locus and dominant lethal mutations in male mice by 1-methyl-1-nitrosourea (MNU)

1-Methyl-1-nitrosourea (MNU) induced specific-locus mutations in mice in all spermatogenic stages except spermatozoa. After intraperitoneal injection of 70 mg/kg body weight of MNU a high yield of specific-locus mutations was observed in spermatids (21.8 × 10⁻⁵ mutations per locus per gamete). The highest mutational yield was induced in differentiating spermatogonia. In 1954 offspring we observed 5 specific-locus mutants (44.8 × 10⁻ mutations per locus per gamete). In addition, 2 mosaics were recovered, which gave a combined mutation rate of 62.7 × 10⁻⁵. In A_s spermatogonia the mutation rate was 3.9 × 10⁻⁵. The same dose of 70 mg/kg of MNU induced dominant lethal mutations 5–48 days post treatment, mainly due to post-implantation loss in spermatids and spermatocytes. It is interesting to compare the induction pattern of mutations by MNU with methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and ethylnitrosourea (ENU). Based on the different spermatogenic response of the induction of specific-locus mutations we can characterize the 4 mutagens in the following way: EMS = MMS ≠ MNU ≠ ENU.     

The frequency of dominant cataract and recessive specific-locus mutations and mutation mosaics in F1 mice derived from post-spermatogonial treatment with ethylnitrosourea

The frequency of dominant cataract and recessive specific-locus mutations and mutation mosaics was determined in F1 mice derived from post-spermatogonial germ-cell stage treatment with 2 X 80, 160 or 250 mg/kg ethylnitrosourea. A total of 5 dominant cataract mutations, 3 dominant cataract mutation mosaics, 1 specific-locus mutation and 9 specific-locus mutation mosaics were recovered in 15,542 screened F1 offspring. Results indicate that ethylnitrosourea treatment increases the mutation rate of dominant cataract and recessive specific-locus alleles in post-spermatogonial germ-cell stages of the mouse and that the mutations occur mainly as mosaics. Genetic confirmation of newly induced mutations occurring as mosaics is more problematical for induced recessive alleles than for induced dominant alleles and should be considered when evaluating such mutagenicity results.     

Enzyme-activity mutations detected in mice after paternal fractionated irradiation

(101/E1 × C3H/E1)F₁-hybrid male mice were exposed in a 24-h fractionation interval to either 3.0 + 3.0-Gy or 5.1 + 5.1-Gy X-irradiation, and mated to untreated Test-stock females. The offspring were examined for mutations at 7 recessive specific loci and for activity alterations of erythrocyte enzymes controlled presumably by 12 loci. No enzyme-activity mutant was found in 3610 F₁-offspring of the control group. In the experimental groups, no mutant was detected in 533 (3.0 + 3.0 Gy) and 173 (5.1 + 5.1 Gy) offspring from postspermatogonial germ cells treated. After treatment of spermatogonia, 1 mutant in 3388 F₁-offspring of the 3.0 + 3.0-Gy group, and 5 mutants in 3187 F₁ offspring of the 5.1 + 5.1-Gy group were found. The mutants were all genetically confirmed. The frequency (expressed as mutants/locus/gamete) of enzyme-activity mutations is 2 (5.1 + 5.1-Gy group) to 10 (3.0 + 3.0-Gy group) times lower than the frequency of recessive specific-locus mutations.     

The frequency of dominant cataract and recessive specific-locus mutations in mice derived from 80 or 160 mg ethylnitrosourea per kg body weight treated spermatogonia

A systematic comparison of the frequency of dominant cataract and recessive specific-locus mutations in mice has been extended to include results for 80 and 160 mg ethylnitrosourea per kg body weight spermatogonial treatment. The frequency of confirmed dominant cataract mutations in the historical control, 80 and 160 mg/kg ethylnitrosourea treatment groups was 1/22594, 8/5090 and 14/6435, respectively. The frequency of recessive specific-locus mutations in the same dose groups was, respectively, 19/227805, 20/13274 and 35/8658. These present results confirm previous results, which indicate that ethylnitrosourea is effective in inducing both recessive specific-locus and dominant cataract mutations although the per locus mutation rate to recessive alleles was observed to be approximately 6 times greater than the per locus mutation rate to dominant alleles. The exclusion of certain classes of lens opacity variant phenotypes, previously demonstrated not to be due to a dominant mutation, from the group of suspected dominant cataract mutations subjected to a genetic confirmation test has greatly improved the efficiency of the test. A total of 23 dominant cataract mutations were confirmed from a group of 67 phenotypic variants. Of the 23 confirmed dominant cataract mutations, 8 were shown to have reduced transmission to the following generation of offspring expressing the mutant phenotype. These results are also consistent with previous results for ethylnitrosourea or radiation treatment in which it was shown that approximately one-third of the recovered mutations have reduced penetrance. One group of dominant cataract mutations, with phenotypic effects on the polar, sub-capsular or corneal regions, is overly represented in the group of recovered mutations with a reduced transmission of offspring expressing the mutant phenotype. Two hypotheses are suggested for this observation, both dependent on the fact that the regions affected indicate that the mutations are expressed later in the development of the eye. Either all carrier individuals have not expressed the phenotype at the time of examination and classification, or later acting mutations are more subject to environmental interactions resulting in more variable expression. Finally, it is argued that a dominant cataract mutation test represents a most practicable protocol to screen for induced dominant mutations in germ cells of the mouse. The imposition of the criterion that suspected variants be subjected to a genetic confirmation test has at least two advantages beside the fact that results represent unambiguous mutational events.(ABSTRACT TRUNCATED AT 400 WORDS)     

Multiple paternity and female-biased mutation at a microsatellite locus in the olive ridley sea turtle (Lepidochelys olivacea)

Multiple paternity in the olive ridley sea turtle (Lepidochelys olivacea) population nesting in Suriname was demonstrated using two microsatellite loci, viz., Ei8 and Cm84. The large number of offspring sampled per clutch (70 on average, ranging from 15 to 103) and the number of alleles found at the two loci (18 and eight alleles, respectively) enabled unambiguous assessment of the occurrence of multiple paternity. In two out of 10 clutches analysed, the offspring had been sired by at least two males, which was confirmed at both loci. In both clutches, unequal paternity occurred: 73% and 92% of the offspring had been sired by the primary male. The probability of detecting multiple paternity was 0.903, and therefore there is a small chance that multiple paternity occurred but remained undetected in some of the eight clutches that appeared to be singly sired. Analysis of 703 offspring revealed a high mutation rate for locus Ei8 (micro = 2.3 x 10(-2)) with all 33 mutations occurring in maternal alleles. In particular, one allele of 274 bp mutated at a high frequency in a clutch to which the mother contributed the allele, but in another clutch where the father contributed the same allele, no such mutations were observed. Inferred allele-specific mutation rates for Ei8 and expected numbers of mutations per clutch confirmed that maternal alleles for Ei8 are more likely to mutate in the olive ridley sea turtle than paternal alleles. Possible explanations are discussed.     

Tests of induction in mice by acute and chronic ionizing radiation and ethylnitrosourea of dominant mutations that cause the more common skeletal anomalies

Assessment-of-Dominant-Damage (ADD) experiments explored induction by proven specific-locus mutagens of dominant mutations that cause skeletal anomalies, cataracts, and stunted growth in offspring of mutagenized male mice. The data set reported here includes 6134 offspring. Mutagenic treatments included 600 R (i.e., approximately 6 Gy) of X-rays delivered in about 7 min, 600 R of gamma rays delivered over about 110 days, and four weekly intraperitoneal (i.p.) injections of 77.5 mg/kg of ethylnitrosourea (ENU). The results reported in this paper are restricted to mutations induced in stem-cell spermatogonia and to the 34 more common skeletal anomalies (i.e., those found in 0.5% or more of the control offspring). Mutation induction was demonstrated for eight anomalies in the acute X-ray experiment and for 17 anomalies (including those same eight anomalies) in the ENU experiment. In spite of the surprisingly high mutation rates found for these treatments, there was no hint of any induction of such dominant mutations by 600 R of chronic gamma radiation. Our results suggest that several anomalies related to variation in the sacralization pattern may be particularly useful for revealing induction of dominant mutations.     

A comparison of the dominant cataract and recessive specific-locus mutation rates induced by treatment of male mice with ethylnitrosourea

Mice were derived from parental males treated with 250 mg ethylnitrosourea per kg body weight. The mice were screened simultaneously for induced dominant cataract and recessive specific-locus mutations. In the spermatogonial treatment group, 16 dominant cataract, 1 dominant corneal opacity and 60 recessive specific-locus mutations were recovered and genetically confirmed in 9352 offspring observed. This lower yield of dominant cataract mutations, when compared with the yield of recessive specific-locus mutations, is similar to results observed by Kratochvilova in a series of experiments on dominant cataract mutations induced by radiation treatment. These results taken with reported results from other dominant mutation test systems, suggest a lower per-locus mutation rate to dominant than to recessive alleles. A corollary to the hypothesis that most dominantly expressed alleles code for an alteration in the function of the normal gene product is that a limited subset of mutations could normally lead to a dominantly expressed mutation. This may explain the lower per-locus mutation rate to dominant than to recessive alleles.

Genetic confirmation tests of recovered presumed dominant cataract mutations indicate that a certain category of phenotypic variants (bilateral, severe or unique lens opacity) is likely to be a true mutation but only represents 7 of the 19 mutations recovered. A second category of phenotypic variants (unilateral, neither severe nor unique lens opacity) has an extremely low probability of being a true mutation. Only 1 confirmed mutation in 181 phenotypic variants was obtained. The remaining category of phenotypic variants (either unilateral severe or unique, or bilateral neither severe nor unique lens opacity) represented the majority, 11, of the confirmed mutations obtained. However, 266 presumed mutations in this category were recovered. If a sub-class of phenotypic variants within this category could be identified that could be ignored owing to a very low probability of being a true mutation, the efficiency of recovery of confirmed dominant cataract mutations would be greatly increased with no sacrifice in the accuracy of the observed mutation rate.

Finally, the 17 confirmed dominant cataract mutations obtained included a class of 7 that produced significantly fewer than the Mendelian expectation of offspring exhibiting the mutant phenotype. This class probably represents both mutations with penetrance effects and mutations with viability effects.

The present experiments represent the first systematic comparison of induced genetically confirmed dominant and recessive mutations for a chemical mutagen in mice. Such results contribute to our limited understanding of the mutation process to dominant alleles.     

Analysis of chromosomal rearrangements induced by postmeiotic mutagenesis with ethylnitrosourea in zebrafish

Mutations identified in zebrafish genetic screens allow the dissection of a wide array of problems in vertebrate biology. Most screens have examined mutations induced by treatment of spermatogonial (premeiotic) cells with the chemical mutagen N-ethyl-N-nitrosourea (ENU). Treatment of postmeiotic gametes with ENU induces specific-locus mutations at a higher rate than premeiotic regimens, suggesting that postmeiotic mutagenesis protocols could be useful in some screening strategies. Whereas there is extensive evidence that ENU induces point mutations in premeiotic cells, the range of mutations induced in postmeiotic zebrafish germ cells has been less thoroughly characterized. Here we report the identification and analysis of five mutations induced by postmeiotic ENU treatment. One mutation, snh(st1), is a translocation involving linkage group (LG) 11 and LG 14. The other four mutations, oep(st2), kny(st3), Df(LG 13)(st4), and cyc(st5), are deletions, ranging in size from less than 3 cM to greater than 20 cM. These results show that germ cell stage is an important determinant of the type of mutations induced. The induction of chromosomal rearrangements may account for the elevated frequency of specific-locus mutations observed after treatment of postmeiotic gametes with ENU.     

Induction of specific-locus mutations in female mice by 1-ethyl-1-nitrosourea and procarbazine

Using the specific-locus method, the ability of 1-ethyl-1-nitrosourea (ENU) and procarbazine hydrochloride (procarbazine) to induce gene mutations in mouse oocytes was tested and confirmed. The sensitive stage for the induction of mutations in oocytes with 160 mg/kg of ENU is 2-4 weeks post treatment. The induced mutation frequency in this mating interval was 5.1 X 10(-7) mutations/locus/gamete/mg/kg. The induction of mutations by procarbazine occurred 8-33 weeks after treatment. The induced mutation frequency in this mating interval for the 400 mg/kg group was 0.4 X 10(-7) mutations/locus/gamete/mg/kg. One third of the induced mutations was lethal in homozygous condition in both experiments. ENU and procarbazine have a lower mutational response in oocytes than in stem-cell spermatogonia.     

Comparison of the genetic effects of equimolar doses of ENU and MNU: while the chemicals differ dramatically in their mutagenicity in stem-cell spermatogonia, both elicit very high mutation rates in differentiating spermatogonia

Mutagenic, reproductive, and toxicity effects of two closely related chemicals, ethylnitrosourea (ENU) and methylnitrosourea (MNU), were compared at equimolar and near-equimolar doses in the mouse specific-locus test in a screen of all stages of spermatogenesis and spermiogenesis. In stem-cell spermatogonia (SG), ENU is more than an order of magnitude more mutagenic than MNU. During post-SG stages, both chemicals exhibit high peaks in mutation yield when differentiating spermatogonia (DG) and preleptotene spermatocytes are exposed. The mutation frequency induced by 75mgMNU/kg during this peak interval is, to date, the highest induced by any single-exposure mutagenic treatment - chemical or radiation - that allows survival of the exposed animal and its germ cells, producing an estimated 10 new mutations per genome. There is thus a vast difference between stem cell and differentiating spermatogonia in their sensitivity to MNU, but little difference between these stages in their sensitivity to ENU. During stages following meiotic metaphase, the highest mutation yield is obtained from exposed spermatids, but for both chemicals, that yield is less than one-quarter that obtained from the peak interval. Large-lesion (LL) mutations were induced only in spermatids. Although only a few of the remaining mutations were analyzed molecularly, there is considerable evidence from recent molecular characterizations of the marker genes and their flanking chromosomal regions that most, if not all, mutations induced during the peak-sensitive period did not involve lesions outside the marked loci. Both ENU and MNU treatments of post-SG stages yielded significant numbers of mutants that were recovered as mosaics, with the proportion being higher for ENU than for MNU. Comparing the chemicals for the endpoints studied and additional ones (e.g., chromosome aberrations, toxicity to germ cells and to animals, teratogenicity) revealed that while MNU is generally more effective, the opposite is true when the target cells are SG.     

Dominant cataract and recessive specific-locus mutations detected in offspring of procarbazine-treated male mice

The induction of dominant cataract mutations by procarbazine was studied concomitantly with the induction of specific-locus mutations in treated male mice. The most effective dose in the specific-locus test, 600 mg/kg of procarbazine, and a fractionated dose of 5 X 200 mg/kg were used. The frequencies of dominant cataract mutations were higher, but not significantly different from the historical control. The ratio between the number of recovered specific-locus and dominant cataract mutations was in accordance with that found in our experiments with gamma-rays (Ehling et al., 1982; Kratochvilova, 1981) or in experiments with ethylnitrosourea (Favor, 1986). A total of 3 dominant cataract mutations were recovered in the offspring of procarbazine-treated spermatogonial stem cells. Two mutations had complete penetrance while the third exhibited a reduced penetrance of approximately 70%. The viability and fertility of the heterozygotes of all 3 mutations were not affected. Only 1 mutation was shown to be viable as a homozygote.     

Bleomycin, unlike other male-mouse mutagens, is most effective in spermatogonia, inducing primarily deletions

Dominant-lethal tests [P.D. Sudman, J.C. Rutledge, J.B. Bishop, W.M. Generoso, Bleomycin: female-specific dominant lethal effects in mice, Mutat. Res. 296 (1992) 205-217] had suggested that Bleomycin sulfate (Blenoxane), BLM, might be a female-specific mutagen. While confirming that BLM is indeed a powerful inducer of dominant-lethal mutations in females that fails to induce such mutations in postspermatogonial stages of males, we have shown in a specific-locus test that BLM is, in fact, mutagenic in males. This mutagenicity, however, is restricted to spermatogonia (stem-cell and differentiating stages), for which the specific-locus mutation rate differed significantly (P<0.008) from the historical control rate. In treated groups, dominant mutations, also, originated only in spermatogonia. With regard to mutation frequencies, this germ-cell-stage pattern is different from that for radiation and for any other chemical studied to date, except ethylnitrosourea (ENU). However, the nature of the spermatogonial specific-locus mutations differentiates BLM from ENU as well, because BLM induced primarily (or, perhaps, exclusively) multilocus deletions. Heretofore, no chemical that induced specific-locus mutations in spermatogonia did not also induce specific-locus as well as dominant-lethal mutations in postspermatogonial stages, making the dominant lethal test, up till now, predictive of male mutagenicity in general. The BLM results now demonstrate that there are chemicals that can induce specific-locus mutations in spermatogonia without testing positive in postspermatogonial stages. Thus, BLM, while not female-specific, is unique, (a) in its germ-cell-stage specificity in males, and (b) in inducing a type of mutation (deletions) that is atypical for the responding germ-cell stages (spermatogonia).     

ENU mutagenesis in the mouse electrophoretic specific-locus test, 1. Dose-response relationship of electrophoretically-detected mutations arising from mouse spermatogonia treated with ethylnitrosourea

The mouse electrophoretic specific-locus test for induced germ-cell mutations, was used to determine the response of spermatogonial stem cells to a series of doses of the germ cell mutagen N-ethyl-N-nitrosourea (ENU). Male DBA/2J and C57B1/6J mice were treated with doses of 50, 100, 200 or 250 mg/kg ENU and their progeny screened for electrophoretically-detectable mutations at 32 separate loci. As expected, increasing doses of ENU led to increasing mutant frequencies. The differences in mutant frequencies between treated DBA/2J and C57B1/6J males were not statistically significant.     

Male-biased mutation rates and the overestimation of extrapair paternity: problem, solution, and illustration using thick-billed murres (Uria lomvia, Alcidae)

The widespread utility of hypervariable loci in genetic studies derives from the high mutation rate, and thus the high polymorphism, of these loci. Recent evidence suggests that mutation rates can be extremely high and may be male biased (occurring in the male germ-line). These two factors combined may result in erroneous overestimates of extrapair paternity, since legitimate offspring with novel alleles will have more mismatches with respect to the biological father than the biological mother. As mutations are male driven, increasing the number of hypervariable loci screened may simply increase the number of mismatches between fathers and their legitimate offspring. Here we describe a simple statistic, the probability of resemblance (PR), to distinguish between mismatches due to parental misassignment versus mutation in either sex or null alleles. We apply this method to parentage data on thick-billed murres (Uria lomvia), and demonstrate that, without considering either mutations or male-biased mutation rates, cases of extrapair paternity (7% in this study) would be grossly overestimated (14.5%-22%). The probability of resemblance can be utilized in parentage studies of any sexually reproducing species when allele or haplotype frequency data are available for putative parents and offspring. We suggest calculating this probability to correctly categorize legitimate offspring when mutations and null alleles may cause mismatches.