Genomic integrity in the male germ line: evidence in support of the disposable soma hypothesis

in Reproduction
Restricted access

The Big Blue λSelect-cII selection system has been employed along with whole-exome sequencing to examine the susceptibility of the male germ line to mutation in two challenging situations (i) exposure to a chemotherapeutic regime including bleomycin, etoposide and cis-platinum (BEP) and (ii) the ageing process. A 3-week exposure to BEP induced complete azoospermia associated with a loss of developing germ cells and extensive vacuolization of Sertoli cell cytoplasm. Following cessation of treatment, spermatozoa first appeared in the caput epididymis after 6 weeks and by 12 weeks motile spermatozoa could be recovered from the cauda, although the count (P < 0.001) and motility (P < 0.01) of these cells were significantly reduced and superoxide generation was significantly elevated (P < 0.001). Despite this increase in free radical generation, no evidence of chromatin instability was detected in these spermatozoa. Furthermore, embryos obtained from females mated at this 12-week time point showed no evidence of an increased mutational load. Similarly, progressive ageing of Big Blue mice had no impact on the quality of the spermatozoa, fertility or mutation frequency in the offspring despite a significant increase in the mutational load carried by somatic tissues such as the liver (P < 0.05). We conclude that the male germ line is highly resistant to mutation in keeping with the disposable soma hypothesis, which posits that genetic integrity in the germ cells will be maintained at the expense of the soma, in light of the former’s sentinel position in safeguarding the stability of the genome.


    Society for Reproduction and Fertility

Article Information


All Time Past Year Past 30 Days
Abstract Views 152 152 54
Full Text Views 730 730 6
PDF Downloads 87 87 7


Related Articles


  • View in gallery

    Histological profile of the testes in BEP-exposed mice during the recovery phase in relation to corresponding controls. Wk represents the numbers of weeks following a 3-week BEP exposure. Images to the left are the controls, images to the right are BEP-exposed animals. Arrows indicate seminiferous tubule sections exhibiting a severe disruption of spermatogenesis in association with extensive vacuolization of the Sertoli cell cytoplasm.

  • View in gallery

    Analysis of BEP-exposed mice during the recovery phase. (A) Percentage of testicular cross sections not revealing evidence of severely disrupted spermatogenesis, (B) testes weights, (C) mean litter size and (D) number of embryonic resorptions at 14 dpc. Open bars are control treatments and closed bars are chemotherapy-treated animals. Probability values inserted into the top right-hand corner of each panel indicates the overall significance due to treatment according to ANOVA. Asterisks at the head of the columns indicate the significance of the difference at each time point. *P < 0.05; **P < 0.01; ***P < 0.001. At least three independent replicates per group.

  • View in gallery

    Sperm quality following exposure to BEP. (A) Number of spermatozoa recovered from the caput epididymis, (B) number of spermatozoa recovered from the cauda epididymis, (C) percentage of motile cells (D) percentage of progressively motile cells, (E) percentage of viable spermatozoa exhibiting high levels of mitochondrial ROS generation by flow cytometry according to the probe MitoSox Red (MSR). (F) Percentage of viable spermatozoa exhibiting a high level of total cellular ROS generation according to the probe dihydroethidium (DHE). Open bars are control treatments and closed bars are chemotherapy treated animals. Probability values inserted into the top right-hand corner of each panel indicates the overall significance due to treatment according to ANOVA. Asterisks at the head of the columns indicate the significance of the difference at each time point. *P < 0.05; **P < 0.01; ***P < 0.001. At least three independent replicates per group.

  • View in gallery

    Levels of DNA damage in the spermatozoa of mice recovering from BEP exposure as measured by SCSA. Results of SCSA analysis for (A) caput epididymal spermatozoa and (B) cauda epididymal spermatozoa. Open bars are control treatments and closed bars are chemotherapy-treated animals. At least three independent replicates per group.

  • View in gallery

    Impact of age on male reproduction in the Big Blue mouse. (A) Number of de novo mutations in the whole-exome of embryos of pairs of young mice (1352 ± 20 mutations) and older mice (1357 ± 9 mutations), showed no statistical differences (Mann–Whitney U test, P > 0.1, n = 4). (B) Mutation frequency in the whole-exome of embryos was not statistically different (Mann–Whitney U test, P > 0.1, n = 4) between the offspring of young (2.98 × 10−7 ± 4 × 10−9 mutation/bp) and aged mice (2.99 × 10−7 ± 2 × 10−9 mutations/bp). Age-dependent changes in (C) testes weight; (D) sperm motility; (E) DNA damage as measured by the SCSA assay; (F) flow cytometry analysis of percentage of cell expressing abnormally high levels of 8OHdG formation; (G) mitochondrial superoxide generation in caput and caudal epididymal spermatozoa; (H) mitochondrial membrane potential in caput and caudal epididymal spermatozoa as measured by JC-1. Probability values inserted into the top right-hand corner of each panel indicates the overall significance of differences due to the source of the spermatozoa (caput or cauda epididymis). Asterisks at the head of the columns indicate the significance of the difference at each time point. *P < 0.05; **P < 0.01; ***P < 0.001. At least three independent replicates per group.


AitkenRJ 2013a Human spermatozoa: revelations on the road to conception. F1000 Prime Reports 5 39. (

AitkenRJ 2013b Age, the environment and our reproductive future: bonking baby boomers and the future of sex. Reproduction 147 S1S11. (

AitkenRJDe IuliisGN 2007 Origins and consequences of DNA damage in male germ cells. Reproductive Biomedicine Online 14 727733. (

AitkenRJSmithTBLordTKuczeraLKoppersAJNaumovskiNConnaughtonHBakerMADe IuliisGN 2013 On methods for the detection of reactive oxygen species generation by human spermatozoa: analysis of the cellular responses to catechol oestrogen, lipid aldehyde, menadione and arachidonic acid. Andrology 1 192205. (

BennettsLEAitkenRJ 2005 A comparative study of oxidative DNA damage in mammalian spermatozoa. Molecular Reproduction and Development 71 7787. (

BieberAMMarconLHalesBFRobaireB. 2006 Effects of chemotherapeutic agents for testicular cancer on the male rat reproductive system, spermatozoa, and fertility. Journal of Andrology 27 189200. (

BiggersJDWhittenWKWhittinghamDG 1971 The culture of mouse embryos in vitro. In Methods in Mammalian Embryology pp 86116. Ed DanielJC. San Francisco: Freeman.

CarielloNFPiegorschWWAdamsWTSkopekTR 1994 Computer program for the analysis of mutational spectra: application to p53 mutants. Carcinogenesis 15 22812285. (

CastilloJSimonLde MateoSLewisSOlivaR 2011 Protamine/DNA ratios and DNA damage in native and density gradient centrifuged sperm from infertile patients. Journal of Andrology 32 324332. (

ChowEJKamineniADalingJRFraserAWigginsCLMineauGPHamreMRSeversonRKDrews-BotschCMuellerBA 2009 Reproductive outcomes in male childhood cancer survivors: a linked cancer-birth registry analysis. Archives of Pediatrics and Adolescent Medicine 163 887894. (

ChoyJTBranniganRE 2013 The determination of reproductive safety in men during and after cancer treatment. Fertility and Sterility 100 11871191. (

CrowJF 2000 The origins, patterns and implications of human spontaneous mutation. Nature Reviews Genetics 1 4047. (

De IuliisGNThomsonK.MitchellLAFinnieJMKoppersAJHedgesANixonBAitkenRJ 2009 DNA damage in human spermatozoa is highly correlated with the efficiency of chromatin remodeling and the formation of 8-hydroxy-2′-deoxyguanosine, a marker of oxidative stress. Biology of Reproduction 81 517524. (

DelbesGHalesBFRobaireB 2007 Effects of the chemotherapy cocktail used to treat testicular cancer on sperm chromatin integrity. Journal of Andrology 28 241249. (

DohleGR 2010 Male infertility in cancer patients: review of the literature. International Journal of Urology 17 327331. (

EvensonDJostL 2000 Sperm chromatin structure assay is useful for fertility assessment. Methods in Cell Science 22 169189. (

HalesBFAguilar-MahechaARobaireB 2005 The stress response in gametes and embryos after paternal chemical exposures. Toxicology and Applied Pharmacology 207 514520. (

HammoudSSNixDAZhangHPurwarJCarrellDTCairnsBR 2009 Distinctive chromatin in human sperm packages genes for embryo development. Nature 460 473478. (

HillKABuettnerVLHalangodaAKunishigeMMooreSRLongmateJScaringeWASommerSS 2004 Spontaneous mutation in Big Blue® mice from fetus to old age: tissue-specific time courses of mutation frequency but similar mutation types. Environmental and Molecular Mutagenesis 43 110120. (

HuangVWLeeCLLeeYLLamKKKoJKYeungWSHoPCChiuPC 2015 Sperm fucosyltransferase-5 mediates spermatozoa-oviductal epithelial cell interaction to protect human spermatozoa from oxidative damage. Molecular Human Reproduction 21 516526. (

JohnsonSLDunleavyJGemmellNJNakagawaS. 2015 Consistent age-dependent declines in human semen quality: a systematic review and meta-analysis. Ageing Research Reviews 19 2233. (

JónssonHSulemPKehrBKristmundsdottirSZinkFHjartarsonEHardarsonMTHjorleifssonKEEggertssonHPGudjonssonSA et al. 2017 Parental influence on human germline de novo mutations in 1548 trios from Iceland. Nature 549 519522. (

KaarouchIBouamoudNMadkourALouanjliNSaadaniBAssouSAboulmaouahibSAmzaziSCopinHBenkhalifaMSefriouiO 2018 Paternal age: negative impact on sperm genome decays and IVF outcomes after 40 years. Molecular Reproduction and Development 85 271280. (

KirkwoodTB 1977 Evolution of ageing. Nature 270 301304. (

KirkwoodTBLAustadAN 2000 Why do we age? Nature 408 233238. (

KongAFriggeMLMassonGBesenbacherSSulemPMagnussonGGudjonssonSASigurdssonAJonasdottirAJonasdottirA et al. 2012 Rate of de novo mutations and the importance of father’s age to disease risk. Nature 488 471475. (

LeonardBHartSNBurnsMBCarpenterMATemizNARathoreAVogelRINikasJBLawEKBrownWL et al. 2013 APOBEC3B upregulation and genomic mutation patterns in serous ovarian carcinoma. Cancer Research 73 72227231. (

LooijengaLHGillisAJStoopHBiermannKOosterhuisJW 2011 Dissecting the molecular pathways of (testicular) germ cell tumour pathogenesis; from initiation to treatment-resistance. International Journal of Andrology 34 e234e251. (

LyonsDMLauringAS 2017 Evidence for the selective basis of transition-to-transversion substitution bias in two RNA viruses. Molecular Biology and Evolution 34 32053215. (

ManochantrSChiamchanyaCSobhonP 2012 Relationship between chromatin condensation, DNA integrity and quality of ejaculated spermatozoa from infertile men. Andrologia 44 187199. (

MarconLHaleBFRobaireB 2008 Reversibility of the effects of subchronic exposure to the cancer chemotherapeutics bleomycin, etoposide, and cisplatin on spermatogenesis, fertility, and progeny outcome in the male rat. Journal of Andrology 29 408417. (

MaselliJHalesBFChanPRobaireB 2012 Exposure to bleomycin, etoposide, and cis-platinum alters rat sperm chromatin integrity and sperm head protein profile. Biology of Reproduction 86 166.

Meeks JJSheinfeld JEggener SE 2012 Environmental toxicology of testicular cancer. Urologic Oncology 30 212215. (

MortensenMSGundgaardMGDaugaardG 2011 Treatment options for carcinoma in situ testis. International Journal of Andrology 34 e32e36. (

NiliHAMozdaraniHAleyasinA 2009 Correlation of sperm DNA damage with protamine deficiency in Iranian subfertile men. Reproductive Biomedicine Online 18 479485. (

NohmiTSuzukiTMasumuraK 2000 Recent advances in the protocols of transgenic mouse mutation assays. Mutation Research 455 191215. (

Pérez-CerezalesSMirandaAGutiérrez-AdánA 2012 Comparison of four methods to evaluate sperm DNA integrity between mouse caput and cauda epididymidis. Asian Journal of Andrology 14 335337.

R Core Team 2004 R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing. (available at:

ReuterVE 2005 Origins and molecular biology of testicular germ cell tumors. Modern Pathology Supplement 2 S51S60. (

SakkasDUrnerFBizzaroDManicardiGBianchiPGShoukirYCampanaA 1998 Sperm nuclear DNA damage and altered chromatin structure: effect on fertilization and embryo development. Human Reproduction Supplement 4 1119. (

SawyerDEMercerBGWiklendtAMAitkenRJ 2003 Quantitative analysis of gene-specific DNA damage in human spermatozoa. Mutation Research 529 2134. (

ShelleyMDBurgonKMasonMD 2002 Treatment of testicular germ-cell cancer: a cochrane evidence-based systematic review. Cancer Treatment Reviews 28 237253. (

SherrySTWardMHKholodovMBakerJPhanLSmigielskiEMSirotkinK 2001 dbSNP: the NCBI database of genetic variation. Nucleic Acids Research 29 308311. (

SignorelloLBMulvihillJJGreenDMMunroHMStovallMWeathersREMertensACWhittonJARobisonLLBoiceJDJr 2012 Congenital anomalies in the children of cancer survivors: a report from the childhood cancer survivor study. Journal of Clinical Oncology 30 239245. (

SkakkebaekNE 1972 Possible carcinoma-in-situ of the testis. Lancet 2 516517. (

StangAJansenLTrabertBRusnerCEberleAKatalinicAEmrichKHolleczekBBrennerH & GEKID Cancer Survival Working Group 2013 Survival after a diagnosis of testicular germ cell cancers in Germany and the United States, 2002–2006: a high resolution study by histology and age. Cancer Epidemiology 37 492497. (

StåhlOBoydHAGiwercmanALindholmMJensenAKjærSKAndersonHCavallin-StåhlERylanderL 2011 Risk of birth abnormalities in the offspring of men with a history of cancer: a cohort study using Danish and Swedish national registries. Journal of the National Cancer Institute 103 398406. (

SweeneyC 2001 History of testicular cancer chemotherapy: maximizing efficacy, minimizing toxicity. Seminars in Urologic Oncology 19 170179.

van BastenJPSchrafford KoopsHSleijferDTPrasEvan DrielMFHoekstraHJ 1997 Current concepts about testicular cancer. European Journal of Surgical Oncology 23 354360. (

YatsenkoANTurekPJ 2018 Reproductive genetics and the aging male. Journal of Assisted Reproduction and Genetics 35 933941. (

ZhanHJiangJSunQKeAHuJHuZZhuKLuoCRenNFanJ et al. 2017 Whole-exome sequencing-based mutational profiling of hepatitis B virus-related early-stage hepatocellular carcinoma. Gastroenterology Research and Practice 2017 2029315. (

ZiglioliFMaestroniUDinaleFCiuffredaMCortelliniP 2011 Carcinoma in situ (CIS) of the testis. Acta Biomedica 82 162169.


Google Scholar