Upstream eventIncrease, Mutations
Increase, Heritable mutations in offspring
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding|
|Alkylation of DNA in male pre-meiotic germ cells leading to heritable mutations||adjacent||High||Moderate|
Life Stage Applicability
Key Event Relationship Description
If a mutation arises in spermatogonial stem cells and does not influence cellular function, the mutation can become fixed and/or propagated within the stem cell population. Mutations that do not affect sperm maturation will persist through the succeeding stages of spermatogenesis and will be found in the mature sperm of the organism throughout its reproductive lifespan. Mutations can also occur in differentiating spermatogonia; however, once the sperm generated by these dividing spermatogonia are ejaculated there will be no ‘record’ of the induced mutation. Mutations that impair spermatogenesis or the viability of the cell will be lost via apoptosis and cell death, potentially contributing to decreased fertility. Mutations that do not impact sperm motility, morphology or ability to penetrate the zona pellucida (or other important sperm parameters), and that are present in mature sperm, may be transmitted to the egg resulting in the development of an offspring with a mutation. Thus, increased incidence of mutations in germ cells leads to increased incidence of mutations in the offspring. There is a great deal of evidence demonstrating that exposure to a variety of DNA alkylating agents induces mutations in male spermatogenic cells.
Evidence Supporting this KER
Evolution requires heritable mutations that are transmitted to offspring via parental gametes. This is the primary mechanism by which an offspring would have a sequence variant in every single one of its cells that is not found in its parents. Indeed, as stated in a recent review in Science by Shendura and Aikey "Germline mutations are the principal cause of heritable disease and the ultimate source of evolutionary change." Thus, this KER is supported by substantive understanding of genetics and evolution, with heritable germ cell mutations forming the basis for the selective evolution of species.
In addition, in toxicology, a large body of literature demonstrates that exposure to specific genotoxic chemicals during pre-meiotic stages of spermatogenesis leads to mutations in both the sperm and the offspring, supporting that mutations occurring in sperm fertilize the egg and result in offspring with mutations (reviewed in Demarini 2012; Marchetti and Wyrobek 2005; Yauk et al. 2012). Indeed, ENU is used as a tool in genetics to create offspring carrying mutations for the purposes of understanding gene function ( e.g., http://www.riken.jp/en/research/labs/brc/mutagen_genom). In these studies, male mice are mutagenized by exposure to ENU and mated to females. The offspring of these males have a much higher incidence of mutation; the function of new mutations occurring in genes in these offspring is used to study gene function.
Thus, overall this KER is biologically plausible and widely understood.
Identification of mutations in sperm requires the destruction of the sperm. Thus, tracking a mutated spermatogonial stem cell through to fertilization and characterization of the mutation in the offspring is not possible, and the empirical evidence to support this KER is weak. No single study has looked at the dose-response relationship of the same mutation endpoint in germ cells and offspring because technologies are not currently available to do this. We caution that comparing mutation rates across different genes or genetic loci is imprecise, because factors intrinsic to specific loci govern mutation rates (e.g., length, GC content, transcribed versus non-transcribed, coding versus non-coding, chromatin structure, DNA methylation, sequence, etc.).
Increased numbers of germ cell mutations occur in mature sperm 42+ days post-exposure in mice (indicating that mutations arose in pre-meiotic male germ cells). Mating in this time interval to produce offspring also results in increased incidence of mutation in the descendants of exposed males, indicating temporal concordance. By virtue of the required experimental designs, mutations measured in the offspring occur after the mutations in germ cells. For example, mutations identified in proteins via electrophoresis (a variation of the SLT test) are found in the offspring of male mice mated 10+ weeks post-exposure to ENU (Lewis et al. 1991). These inherited changes are the result of mutations in stem cells that persist through spermatogenesis and are transmitted to offspring.
The only assay that presently can measure mutations in both sperm and offspring is the tandem repeat mutation assay. A single study on one dose of radiation (1 Gy X-ray) against matched controls has shown that increases in mutation frequencies in exposed sperm are similar to the increases observed in the offspring of exposed males for tandem repeats (Yauk et al. 2002), suggesting that tandem repeat mutations in sperm are transmitted to offspring. Alkylating agents cause similar increases in tandem repeat mutations in both sperm and in the offspring through comparison across studies (Dubrova et al. 2008; Swayne et al. 2012; Vilarino-Guell et al. 2003), but dose-response studies have not been conducted. It is advisable that dose-response experiments in sperm for tandem repeats be conducted in the future to address this gap.
Many studies have shown the induction of transgene mutations recovered in mature sperm derived from toxicant-exposed pre-meiotic male germ cells in transgenic mutation reporter mice (e.g., Brooks and Dean 1997; Liegibel and Schmezer 1997; Mattison et al. 1997). One study measured transgene mutations in the offspring of mice exposed to three single i.p. doses of 100 mg/kg ENU (in 7 day intervals) (Barnett et al. 2002). Four inherited transgene mutations were found from 280 mice (confirmed in multiple somatic tissues), for a mutant frequency of 35.7 x 10E-5 per locus. There is no comparable study on the sperm of lacI transgenic mice, or a similar exposure in another transgenic strain for comparison. However, conversion of the per locus lacI offspring mutant frequency to per nucleotide reveals a mutant frequency of 3.31 x 10-7 per nucleotide. LacZ mutant frequencies for 150 mg/kg (half the exposure level of the lacI transgenic mice) exposures of MutaMouse males to ENU results in a per nucleotide mutation frequency ranging from 0.37 to 2.21 x 10E-7 (Liegibel and Schmezer 1997; van Delft and Baan 1995). Thus, the induced mutation frequency in sperm and offspring are within the same range, despite higher doses in the offspring study, supporting incidence concordance for these events.
Finally, it has been documented that DNA damage and mutation accumulates as human males age (reviewed in Paul and Robaire 2013), which is concordant with increased incidence of mutation in the offspring of ageing fathers (Kong et al. 2012; Sun et al. 2012). Comparison of the dose-response characteristics of this relationship is not possible because of differences in the mutagenic endpoints measured in sperm versus offspring.
Uncertainties and Inconsistencies
There are no inconsistencies in the data for this KER, although the data are limited. There is a possibility that mutations can arise in the early embryo instead of in the spermatogenic cells. However, given the study designs for this type of work (where > 42 days pass prior to sperm collection or mating – see OECD TG488 for the time-series required for transgene mutation analysis in sperm), it is unlikely that this contributes significantly. Limitations in technology currently prevent the analyses required to describe the incidence of mutations in sperm versus offspring, but this is a future research direction. It should be noted that the locations and types of mutations would influence the probably of transmission; this relationship has not been confirmed empirically and limits extrapolation across studies applying different endpoints.
Quantitative Understanding of the Linkage
Mutations conferring a selective disadvantage to sperm or to the embryo will not be measured in live born offspring and will be eliminated. Thus, mutation frequency in sperm should be equal to mutation rate derived by measuring mutations in the offspring for non-selective loci (as is seen in the rodent tandem repeat and transgene mutation examples described above); or, sperm mutation frequency should be greater than mutation rate measured by identifying mutations in the offspring. However, quantitative data to demonstrate this are lacking because of current technical limitations to study this. It is anticipated that improved models will be developed to predict the likely outcome of increased rates of heritable mutation from sperm mutation frequency data when more data are available from studies applying next generation sequencing technologies in sperm and pedigrees.
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
Mutation is the underlying source of evolution and occurs in every species. Theoretically, any sexually reproducing organism (i.e., producing gametes) can acquire mutations in their gametes and transmit these to descendants. Thus, the present KER is relevant to any species producing sperm.
Barnett, L.B., R.W. Tyl, B.S. Shane, M.D. Shelby and S.E. Lewis (2002), "Transmission of mutations in the lacI transgene to the offspring of ENU-treated Big Blue male mice", Environ. Mol. Mutagen., 40(4): 251-257.
Brooks, T.M. and S.W. Dean (1997), "The detection of gene mutation in the tubular sperm of Muta Mice following a single intraperitoneal treatment with methyl methanesulphonate or ethylnitrosourea", Mutat. Res., 388(2-3): 219-222.
Demarini, D.M. (2012), "Declaring the existence of human germ-cell mutagens", Environ. Mol. Mutagen., 53(3): 166-172.
Dubrova, Y.E., P. Hickenbotham, C.D. Glen, K. Monger, H.P. Wong and R.C. Barber (2008), "Paternal exposure to ethylnitrosourea results in transgenerational genomic instability in mice", Environ. Mol. Mutagen., 49(4): 308-311.
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Lewis, S.E., L.B. Barnett, B.M. Sadler and M.D. Shelby (1991), "ENU mutagenesis in the mouse electrophoretic specific-locus test, 1. Dose-response relationship of electrophoretically-detected mutations arising from mouse spermatogonia treated with ethylnitrosourea", 'Mutat. Res., 249(2): 311-315.
Liegibel, U.M. and P. Schmezer (1994), "Detection of the two germ cell mutagens ENU and iPMS using the LacZ/transgenic mouse mutation assay" Mutat. Res., 341(1):17-28.
Marchetti, F. and A.J. Wyrobek (2005), "Mechanisms and consequences of paternally-transmitted chromosomal abnormalities", Birth Defects Res C Embryo Today, 75(2): 112-129.
Mattison, J.D., L.B. Penrose and B. Burlinson (1997), "Preliminary results of ethylnitrosourea, isopropyl methanesulphonate and methyl methanesulphonate activity in the testis and epididymal spermatozoa of Muta Mice", Mutat. Res. 388(2-3): 123-7.
O'Brien, J.M., A. Williams, J. Gingerich, G.R. Douglas, F. Marchetti and C.L. Yauk (2013), "No evidence for transgenerational genomic instability in the F1 or F2 descendants of Muta™Mouse males exposed to N-ethyl-N-nitrosourea", Mutat Res., 741-742:11-7
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Shendura, J. and M. Akey (2015), "The origins, determinants, and consequences of human mutations", Science, 349(6255): 1478-1483.
Sun, J.X., A. Helgason, G. Masson, S.S. Ebenesersdottir, H. Li, S. Mallick, S. Gnerre, N. Patterson, A. Kong, D. Reich and K. Stefansson (2012), "A direct characterization of human mutation based on microsatellites", Nat. Genet., 44(10): 1161-1165.
Swayne, B.G., A. Kawata, N.A. Behan, A. Williams, M.G. Wade, A.J. Macfarlane and C.L. Yauk (2012), "Investigating the effects of dietary folic acid on sperm count, DNA damage and mutation in Balb/c mice", Mutat. Res., 737(1-2): 1-7.
Vilarino-Guell, C., A.G. Smith and Y.E. Dubrova (2003), "Germline mutation induction at mouse repeat DNA loci by chemical mutagens", 'Mutat. Res., 526(1-2): 63-73.
Yauk, C.L., Y.E. Dubrova, G.R. Grant and A.J. Jeffreys (2002), "A novel single molecule analysis of spontaneous and radiation-induced mutation at a mouse tandem repeat locus", Mutat Res., 500(1-2): 147-156.
Yauk, C.L., L.J. Argueso, S.S. Auerbach, P. Awadalla, S.R. Davis, D.M. Demarini, G.R. Douglas, Y.E. Dubrova, R.K. Elespuru, T.M. Glover, B.F. Hales , M.E. Hurles, C.B. Klein, J.R. Lupski, D.K. Manchester, F. Marchetti, A. Montpetit, J.J. Mulvihill, B. Robaire, W.A. Robbins, G.A. Rouleau, D.T. Shaughnessy, C.M. Somers, J.G. Taylor 6th, J. Trasler, M.D. Waters, T.E. Wilson, K.L. Witt and J.B. Bishop (2013), "Harnessing genomics to identify environmental determinants of heritable disease" Mutation Research, 752(1): 6-9.