Molecular Biology Reports

, Volume 40, Issue 8, pp 4747–4757 | Cite as

Aberrant DNA methylation at Igf2–H19 imprinting control region in spermatozoa upon neonatal exposure to bisphenol A and its association with post implantation loss

  • Tanvi Doshi
  • Criselle D’souza
  • Geeta VanageEmail author


Bisphenol A (BPA) is an estrogenic compound commonly used in manufacture of various consumer products. Earlier studies from our group have demonstrated that neonatal exposure of male rats to BPA causes decrease in sperm count and motility, increase in post implantation loss, ultimately leading to subfertility during adulthood. One of the factors contributing for post implantation loss is altered methylation pattern of imprinted genes. The present study was undertaken to investigate the molecular effects of neonatal exposure of male rats to BPA (2.4 μg/pup) (F0) on the methylation of H19 imprinting control region (ICR) in resorbed embryo (F1) and compared with spermatozoa of their respective sires (F0). We observed a significant down regulation in the transcript expression of Igf2 and H19 genes in BPA resorbed embryo (F1) as compared to control viable embryo. A significant hypomethylation was observed at the H19 ICR in the spermatozoa as well as in resorbed embryo sired by rats exposed neonatally to BPA. These results indicated that the aberrant methylation at ICR in spermatozoa was inherited by embryo which causes perturbation in the expression of Igf2 and H19, ultimately leading to post implantation loss. This could be one of the possible mechanisms of BPA induced adverse epigenetic effects on male fertility.


Bisphenol A DNA methylation Imprinting control region Post implantation loss Neonatal exposure 



This work was supported by the Department of Science and Technology (Grant numbers: 100/IFD/6122/2010–2011). The author would like to acknowledge the Indian Council of Medical Research for fellowship (Grant number: FNO-3/1/2/6/10-RCH) to Ms. Tanvi Doshi. The authors are grateful to Dr. Dighe for his help in primer designing and Dr. A. Maitra for her guidance with DNA sequencing, Mr. Saravanan and Ms. Nanda for technical assistance with DNA sequencing. The author would like to acknowledge the technical and animal experimentation assistance of Mr. S. Bhagat, Mr. D. Tiwari, Mr. S. Kadam, Mr. P. Salunke, Mr. J. Tare and Mr. M. Mali.

Conflict of interest

The authors declare that there is no potential conflict of interest amongst them.

Supplementary material

11033_2013_2571_MOESM1_ESM.xls (31 kb)
Supplementary material 1 (XLS 31 kb)


  1. 1.
    Li E (2002) Chromatin modification and epigenetic reprogramming in mammalian development. Nat Rev Genet 3:662–673PubMedCrossRefGoogle Scholar
  2. 2.
    Holmes R, Soloway PD (2006) Regulation of imprinted DNA methylation. Cytogenet Genome Res 113:122–129PubMedCrossRefGoogle Scholar
  3. 3.
    Bartolomei M (2009) Genomic imprinting: employing and avoiding epigenetic processes. Genes Dev 15:2124–2133CrossRefGoogle Scholar
  4. 4.
    DeChiara T, Robertson E, Efstratiadis A (1991) Parental imprinting of the mouse insulin-like growth factor II gene. Cell 22:849–859CrossRefGoogle Scholar
  5. 5.
    Kaffer C, Grinberg A, Pfeifer K (2001) Regulatory mechanisms at the mouse Igf2/H19 locus. Mol Cell Biol 21:8189–8196PubMedCrossRefGoogle Scholar
  6. 6.
    Singal R, Ginder G (1999) DNA methylation. Blood 9:4059–4070Google Scholar
  7. 7.
    Santos F, Hendrich B, Reik W, Dean W (2002) Dynamic reprogramming of DNA methylation in the early mouse embryo. Dev Biol 241:172–182PubMedCrossRefGoogle Scholar
  8. 8.
    Santos F, Zakhartchenko V, Stojkovic M, Peters A, Jenuwein T, Wolf E, Reik W, Dean W (2003) Epigenetic marking correlates with developmental potential in cloned bovine preimplantation embryos. Curr Biol 13:1116–1121PubMedCrossRefGoogle Scholar
  9. 9.
    Dolinoy DC, Jirtle RL (2008) Environmental epigenomics in human health and disease. Environ Mol Mutagen 49:4–8PubMedCrossRefGoogle Scholar
  10. 10.
    Kishigami S, Van Thuan N, Hikichi T, Ohta H, Wakayama S, Mizutani E, Wakayama T (2006) Epigenetic abnormalities of the mouse paternal zygotic genome associated with microinsemination of round spermatids. Dev Biol 289:195–205PubMedCrossRefGoogle Scholar
  11. 11.
    Singh R, Kelsey A, Agarwal A (2011) Epigenetics spermatogenesis and male infertility. Mutat Res 727:62–71CrossRefGoogle Scholar
  12. 12.
    Kobayashi H, Sato A, Otsu E, Hiura H, Tomatsu C, Utsunomiya T, Sasaki H, Yaegashi N, Arima T (2007) Aberrant DNA methylation of imprinted loci in sperm from oligospermic patients. Hum Mol Genet 16:2542–2551PubMedCrossRefGoogle Scholar
  13. 13.
    Marques CJ, Costa P, Vaz B, Carvalho F, Fernandes S, Barros A, Sousa M (2008) Abnormal methylation of imprinted genes in human sperm is associated with oligozoospermia. Mol Hum Reprod 14:67–74PubMedCrossRefGoogle Scholar
  14. 14.
    Doerksen T, Benoit G, Trasler JM (2000) Deoxyribonucleic acid hypomethylation of male germ cells by mitotic and meiotic exposure to 5-azacytidine is associated with altered testicular histology. Endocrinology 141:3235–3244PubMedCrossRefGoogle Scholar
  15. 15.
    Anway M, Cupp A, Uzumcu M, Skinner M (2005) Epigenetic transgenerational actions of endocrine disruptors and male fertility. Science 308:1466–1469PubMedCrossRefGoogle Scholar
  16. 16.
    Pathak S, Kedia-Mokashi N, Saxena M, D’Souza R, Maitra A, Parte P, Gill-Sharma M, Balasinor N (2009) Effect of tamoxifen treatment on global and insulin-like growth factor 2–H19 locus-specific DNA methylation in rat spermatozoa and its association with embryo loss. Fertil Steril 91:2253–2263PubMedCrossRefGoogle Scholar
  17. 17.
    Pathak S, Saxena M, D’Souza R, Balasinor N (2010) Disrupted imprinting status at the H19 differentially methylated region is associated with the resorbed embryo phenotype in rats. Reprod Fertil Dev 22:939–948PubMedCrossRefGoogle Scholar
  18. 18.
    Brotons J, Olea-Serrano M, Villalobos M, Pedraza V, Olea N (1995) Xenoestrogens released from lacquer coatings in food cans. Environ Health Perspect 103:608–612PubMedCrossRefGoogle Scholar
  19. 19.
    Olea N, Pulgar R, Pérez P, Olea-Serrano F, Rivas A, Novillo-Fertrell A, Pedraza V, Soto A, Sonnenschein C (1996) Estrogenicity of resin-based composites and sealants used in dentistry. Environ Health Perspect 104:298–305PubMedCrossRefGoogle Scholar
  20. 20.
    Vandenberg L, Chahoud I, Heindel J, Padmanabhan V, Paumgartten F, Schoenfelder G (2010) Urinary, circulating and tissue biomonitoring studies indicate widespread exposure to bisphenol A. Environ Health Perspect 118:1055–1070PubMedCrossRefGoogle Scholar
  21. 21.
    Vandenberg L, Hauser R, Marcus M, Olea N, Welshons W (2007) Human exposure to bisphenol A BPA. Reprod Toxicol 24:139–177PubMedCrossRefGoogle Scholar
  22. 22.
    Markey CM, Luque EH, Muñoz de Toro M, Sonnenschein C, Soto AM (2001) In utero exposure to bisphenol A alters the development and tissue organization of the mouse mammary gland. Biol Reprod 65:1215–1223PubMedGoogle Scholar
  23. 23.
    Ho SM, Tang WY, Belmonte de, Frausto J, Prins GS (2006) Developmental exposure to estradiol and bisphenol A increases susceptibility to prostate carcinogenesis and epigenetically regulates phosphodiesterase type 4 variant 4. Cancer Res 66:5624–5632PubMedCrossRefGoogle Scholar
  24. 24.
    Kandaraki E, Chatzigeorgiou A, Livadas S, Palioura E, Economou F, Koutsilieris M, Palimeri S, Panidis D, Diamanti-Kandarakis E (2011) Endocrine disruptors and polycystic ovary syndrome PCOS: elevated serum levels of bisphenol A in women with PCOS. J Clin Endocrinol Metab 96:E480–E484PubMedCrossRefGoogle Scholar
  25. 25.
    Sugiura-Ogasawara M, Ozaki Y, Sonta S, Makino T, Suzumori K (2005) Exposure to bisphenol A is associated with recurrent miscarriage. Hum Reprod 20:2325–2329PubMedCrossRefGoogle Scholar
  26. 26.
    Li DK, Zhou Z, Miao M, He Y, Wang J, Ferber J, Herrinton LJ, Gao E, Yuan W (2010) Urine bisphenol-A (BPA) level in relation to semen quality. Fertil Steril 95:625–630PubMedCrossRefGoogle Scholar
  27. 27.
    Goyal HO, Robateau A, Braden TD, Williams CS, Srivastava KK, Ali K (2003) Neonatal estrogen exposure of male rats alters reproductive functions at adulthood. Biol Reprod 68:2081–2091PubMedCrossRefGoogle Scholar
  28. 28.
    Sharpe RM, Atanassova N, McKinnell C, Parte P, Turner KJ, Fisher JS, Kerr JB, Groome NP, Macpherson S, Millar MR, Saunders PK (1998) Abnormalities in functional development of the Sertoli cells in rats treated neonatally with diethylstilbestrol: a possible role for estrogens in Sertoli cell development. Biol Reprod 59:1084–1094PubMedCrossRefGoogle Scholar
  29. 29.
    Toyama Y, Yuasa S (2004) Effects of neonatal administration of 17beta-estradiolbeta-estradiol 3-benzoate or bisphenol A on mouse and rat spermatogenesis. Reprod Toxicol 191:81–88Google Scholar
  30. 30.
    Salian S, Doshi T, Vanage G (2009) Neonatal exposure of male rats to bisphenol A impairs fertility and expression of sertoli cell junctional proteins in the testis. Toxicology 12:56–67CrossRefGoogle Scholar
  31. 31.
    Baker J, Liu J, Robertson E, Efstratiadis A (1993) Role of insulin-like growth factors in embryonic and postnatal growth. Cell 75:73–82PubMedGoogle Scholar
  32. 32.
    Taylor JA, Welshons WV, vom Saal FS (2008) No effect of route of exposure (oral; subcutaneous injection) on plasma bisphenol A throughout 24 h after administration in neonatal female mice. Reprod Toxicol 25:169–176PubMedCrossRefGoogle Scholar
  33. 33.
    Doshi T, Mehta SS, Dighe V, Balasinor N, Vanage G (2011) Hypermethylation of estrogen receptor promoter region in adult testis of rats exposed neonatally to bisphenol A. Toxicology 289:74–82PubMedCrossRefGoogle Scholar
  34. 34.
    Livak K, Schmittgen T (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(–Delta Delta C(T)) method. Methods 25:402–408PubMedCrossRefGoogle Scholar
  35. 35.
    Bustin S, Benes V, Garson J, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl M, Shipley G, Vandesompele J, Wittwer C (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622PubMedCrossRefGoogle Scholar
  36. 36.
    Zhu B, Huang X, Chen J, Lu Y, Chen Y, Zhao J (2006) Methylation changes of H19 gene in sperms of X-irradiated mouse and maintenance in offspring. Biochem Biophys Res Commun 340:83–89PubMedCrossRefGoogle Scholar
  37. 37.
    vom Saal FS, Hughes C (2005) An extensive new literature concerning low-dose effects of bisphenol A shows the need for a new risk assessment. Environ Health Perspect 113:926–933CrossRefGoogle Scholar
  38. 38.
    Aikawa H, Koyama S, Matsuda M, Nakahashi K, Akazome Y, Mori T (2004) Relief effect of vitamin A on the decreased motility of sperm and the increased incidence of malformed sperm in mice exposed neonatally to Bisphenol A. Cell Tissue Res 315:119–124PubMedCrossRefGoogle Scholar
  39. 39.
    vom Saal F, Cooke P, Buchanan D, Palanza P, Thayer K, Nagel S, Parmigiani S, Welshons W (1998) A physiologically based approach to the study of bisphenol A and other estrogenic chemicals on the size of reproductive organs daily sperm production and behavior. Toxicol Ind Health 14:239–260Google Scholar
  40. 40.
    Dolinoy D, Huang D, Jirtle R (2007) Maternal nutrient supplementation counteracts bisphenol A-induced DNA hypomethylation in early development. Proc Natl Acad Sci USA 32:13056–13061CrossRefGoogle Scholar
  41. 41.
    Yaoi T, Itoh K, Nakamura K, Ogi H, Fujiwara Y, Fushiki S (2008) Genome-wide analysis of epigenomic alterations in fetal mouse forebrain after exposure to low doses of bisphenol A. Biochem Biophys Res Commun 21:563–567CrossRefGoogle Scholar
  42. 42.
    Bromer J, Zhou Y, Taylor M, Doherty L, Taylor H (2010) Bisphenol-A exposure in utero leads to epigenetic alterations in the developmental programming of uterine estrogen response. FASEB J 24:2273–2280PubMedCrossRefGoogle Scholar
  43. 43.
    Pathak S, D’Souza R, Ankolkar M, Gaonkar R, Balasinor NH (2010) Potential role of estrogen in regulation of the insulin-like growth factor2-H19 locus in the rat testis. Mol Cell Endocrinol 15:110–117CrossRefGoogle Scholar
  44. 44.
    Kobayashi H, Hiura H, John RM, Sato A, Otsu E, Kobayashi N, Suzuki R, Suzuki F, Hayashi C, Utsunomiya T, Yaegashi N, Arima T (2009) DNA methylation errors at imprinted loci after assisted conception originate in the parental sperm. Eur J Hum Genet 171:582–591Google Scholar
  45. 45.
    Brown K, Robertson K (2007) DNMT1 knockout delivers a strong blow to genome stability and cell viability. Nat Genet 39:289–290PubMedCrossRefGoogle Scholar
  46. 46.
    Okano M, Bell DW, Haber DA, Li E (1999) DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99:247–257PubMedCrossRefGoogle Scholar
  47. 47.
    Doshi T, D’Souza C, Dighe V, Vanage G (2012) Effect of neonatal exposure on male rats to bisphenol A on the expression of DNA methylation machinery in the post implantation embryo. J Biochem Mol Toxicol. doi: 10.1002/jbt.21425 PubMedGoogle Scholar
  48. 48.
    Chao H, Zhang X, Chen B, Pan B, Zhang L, Li L, Sun X, Shi Q, Shen W (2012) Bisphenol A exposure modifies methylation of imprinted genes in mouse oocytes via the estrogen receptor signaling pathway. Histochem Cell Biol 137:249–259PubMedCrossRefGoogle Scholar
  49. 49.
    Murphy SK, Zuang H, Wen Y, Spillman M, Whitaker RS, Simel LR, Nichols TD, Marks JR, Berchuck A (2006) Frequent IGF2/H19 domain epigenetic alterations and elevated IGF2 expression in epithelial ovarian cancer. Mol Cancer Res 4:283–292PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  1. 1.National Center for Preclinical Reproductive and Genetic ToxicologyNational Institute for Research in Reproductive Health (ICMR)MumbaiIndia

Personalised recommendations