title: Damaging legacy: maternal cigarette smoking has long-term consequences for male offspring fertility.
abstract: OBJECTIVE
  What are the effects on fertility of cigarette smoke-induced toxicity on male offspring exposed during the gestational/weaning period?
  
  
  CONCLUSIONS
  Maternal cigarette smoke exposure during the gestational/weaning period causes long-term defects in male offspring fertility.
  
  
  BACKGROUND
  Cigarette smoke is a well-known reproductive toxicant which is particularly harmful to both fetal and neonatal germ cells. However, recent studies suggest a significant portion of young mothers in the developed world still smoke during pregnancy. In the context of male reproductive health, our understanding of the effects of in utero exposure on offspring fertility is limited.
  
  
  METHODS
  In this study, 27 C57BL/6 5-week-old female mice were exposed via the nose-only to cigarette smoke (treatment) or 27 were exposed to room air (control) for 6 weeks before being housed with stud males to produce litters. In the treatment group, smoke exposure continued throughout mating, pregnancy and lactation until weaning of pups at 21 days post birth. Male offspring were examined at post-natal days 3, 6, 12, 21 and 98 (adult).
  
  
  METHODS
  Approximately 108 maternal smoke-exposed C57BL/6 offspring and controls were examined. Spermatogenesis was examined using testicular histology and apoptosis/DNA damage was assessed using caspase immunohistochemistry and TUNEL. Sertoli cell morphology and fluctuations in the spermatogonial stem cell population were also examined using immunohistochemistry. Microarray and QPCR analysis were performed on adult testes to examine specific long-term transcriptomic alteration as a consequence of maternal smoke exposure. Sperm counts and motility, zona/oolemma binding assays, COMET analysis and mitochondrial genomic sequencing were also performed on spermatozoa obtained from adult treated and control mice. Fertility trials using exposed adult male offspring were also performed.
  
  
  RESULTS
  Maternal cigarette smoke exposure caused increased gonocyte and meiotic spermatocyte apoptosis (P < 0.01) as well as germ cell depletion in the seminiferous tubules of neonatal and juvenile offspring. Aberrant testicular development characterized by abnormal Sertoli and germ cell organization, a depleted spermatogonial stem cell population (P < 0.01), atrophic seminiferous tubules and increased germ cell DNA damage (P < 0.01) persisted in adult offspring 11 weeks after exposure. Microarray analysis of adult offspring testes associated these defects with meiotic germ cell development, sex hormone metabolism, oxidative stress and Sertoli cell signalling. Next generation sequencing also revealed a high mitochondrial DNA mutational load in the testes of adult offspring (P < 0.01). Adult maternal smoke-exposed offspring also had reduced sperm counts with spermatozoa exhibiting morphological abnormalities (P < 0.01), affecting motility and fertilization potential. Odf2, a spermatozoa flagellum component required for coordinated ciliary beating, was also significantly down-regulated (P < 0.01) in maternal smoke-exposed adult offspring, with aberrant localization along the spermatozoa flagellum. Adult maternal smoke-exposed offspring took significantly longer to impregnate control females and had a slight but significant (P < 0.01) reduction in litter size.
  
  
  CONCLUSIONS
  This study examined only one species (mouse) using a smoking model which only simulates human cigarette smoke exposure.
  
  
  CONCLUSIONS
  This study represents the first comprehensive animal model of maternal smoking on male offspring reproductive function, suggesting that exposure during the gestational/weaning period causes long-term defects in male offspring fertility. This is due to a compromised spermatogonial stem cell population resulting from gonocyte apoptosis and impaired spermatogenic development. This results in significant germ cell damage and Sertoli cell dysfunction, impacting germ cell number, tubule organization, DNA damage and spermatozoa in adult offspring. This study strengthens the current literature suggesting that maternal exposure impairs male offspring fertility, which is currently debated due to conflicting studies.
  
  
  BACKGROUND
  This study was funded by the Australian Research Council, Hunter Medical Research Institute, National Health and Medical Research Council of Australia and the Newcastle Permanent Building Society Charitable Trust. The authors declare no conflict of interest.
authors.affiliation: Reproductive Science Group, School of Environmental & Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia Priority Research Centre for Chemical Biology, University of Newcastle, Callaghan, NSW 2308, Australia.
authors.forename: A P
authors.initials: AP
authors.lastname: Sobinoff
authors.affiliation: Reproductive Science Group, School of Environmental & Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia Priority Research Centre for Chemical Biology, University of Newcastle, Callaghan, NSW 2308, Australia.
authors.forename: J M
authors.initials: JM
authors.lastname: Sutherland
authors.affiliation: Centre for Asthma and Respiratory Disease, University of Newcastle and Hunter Medical Research Institute, Newcastle, Callaghan, NSW 2308, Australia.
authors.forename: E L
authors.initials: EL
authors.lastname: Beckett
authors.affiliation: Reproductive Science Group, School of Environmental & Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia Priority Research Centre for Chemical Biology, University of Newcastle, Callaghan, NSW 2308, Australia.
authors.forename: S J
authors.initials: SJ
authors.lastname: Stanger
authors.affiliation: Centre for Asthma and Respiratory Disease, University of Newcastle and Hunter Medical Research Institute, Newcastle, Callaghan, NSW 2308, Australia.
authors.forename: R
authors.initials: R
authors.lastname: Johnson
authors.affiliation: Centre for Asthma and Respiratory Disease, University of Newcastle and Hunter Medical Research Institute, Newcastle, Callaghan, NSW 2308, Australia.
authors.forename: A G
authors.initials: AG
authors.lastname: Jarnicki
authors.affiliation: Priority Research Centre for Chemical Biology, University of Newcastle, Callaghan, NSW 2308, Australia.
authors.forename: A
authors.initials: A
authors.lastname: McCluskey
authors.affiliation: Centre for Genetic Diseases, MIMR-PHI Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton Vic 3168, Australia.
authors.forename: J C
authors.initials: JC
authors.lastname: St John
authors.affiliation: Centre for Asthma and Respiratory Disease, University of Newcastle and Hunter Medical Research Institute, Newcastle, Callaghan, NSW 2308, Australia.
authors.forename: P M
authors.initials: PM
authors.lastname: Hansbro
authors.affiliation: Reproductive Science Group, School of Environmental & Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia Priority Research Centre for Chemical Biology, University of Newcastle, Callaghan, NSW 2308, Australia Monash Medical Centre, Monash Institute of Medical Research, Clayton, VIC 3168, Australia eileen.mclaughlin@newcastle.edu.au.
authors.forename: E A
authors.initials: EA
authors.lastname: McLaughlin
mesh_terms: D000818:Animals; D017209:Apoptosis; D004249:DNA Damage; D005260:Female; D007248:Infertility, Male / Q000209:etiology*; D007774:Lactation; D008297:Male; D008810:Mice, Inbred C57BL; D011247:Pregnancy; D011297:Prenatal Exposure Delayed Effects; D012708:Sertoli Cells / Q000166:cytology; D012907:Smoking / Q000009:adverse effects*; D013091:Spermatogenesis
keywords: Cigarette smoking; DNA damage; spermatogenesis; subfertility; testis