Heat Stress Causes Aberrant DNA Methylation of H19 and lgf-2r in Mouse Blastocysts

  • Zhu, Jia-Qiao (Gansu Agricultural University) ;
  • Liu, Jing-He (State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Science) ;
  • Liang, Xing-Wei (State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Science) ;
  • Xu, Bao-Zeng (State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Science) ;
  • Hou, Yi (State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Science) ;
  • Zhao, Xing-Xu (Gansu Agricultural University) ;
  • Sun, Qing-Yuan (State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Science)
  • Received : 2007.07.06
  • Accepted : 2007.10.29
  • Published : 2008.04.30


To gain a better understanding of the methylation imprinting changes associated with heat stress in early development, we used bisulfite sequencing and bisulfite restriction analysis to examine the DNA methylation status of imprinted genes in early embryos (blastocysts). The paternal imprinted genes, H19 and Igf-2r, had lower methylation levels in heat-stressed embryos than in control embryos, whereas the maternal imprinted genes, Peg3 and Peg1, had similar methylation pattern in heat-stressed embryos and in control embryos. Our results indicate that heat stress may induce aberrant methylation imprinting, which results in developmental failure of mouse embryos, and that the effects of heat shock on methylation imprinting may be gene-specific.


Supported by : National Natural Science Foundation of China, Chinese Academy of Sciences


  1. Kafri, T., Ariel, M., Brandeis, M., Shemer, R., Urven, L., McCarrey, J., Cedar, H., and Razin, A. (1992). Developmental pattern of gene-specific DNA methylation in the mouse embryo and germ line. Genes Dev. 6, 705-714 https://doi.org/10.1101/gad.6.5.705
  2. Liu, J.H., Yin, S., Xiong, B., Hou, Y., Chen, D.Y., and Sun, Q.Y. (2008). Aberrant DNA methylation imprints in aborted bovine clones. Mol. Reprod. Dev. 75, 598-607 https://doi.org/10.1002/mrd.20803
  3. Lucifero, D., Mertineit, C., Clarke, H.J., Bestor, T.H., and Trasler, J.M. (2002). Methylation dynamics of imprinted genes in mouse germ cells. Genomics 79, 530-538 https://doi.org/10.1006/geno.2002.6732
  4. Matsuzuka, T., Ozawa, M., Nakamura, A., Ushitani, A., Hirabayashi, M., and Kanai, Y. (2005). Effects of heat stress on the redox status in the oviduct and early embryonic development in mice. J. Reprod. Dev. 51, 281-287 https://doi.org/10.1262/jrd.16089
  5. Monk, M., Boubelik, M., and Lehnert, S. (1987). Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development. Development 99, 371-382
  6. Olek, A., and Walter, J. (1997). The pre-implantation ontogeny of the H19 methylation imprint. Nat. Genet 17, 275-276 https://doi.org/10.1038/ng1197-275
  7. Sanford, J.P., Clark, H.J., Chapman, V.M., and Rossant, J. (1987). Differences in DNA methylation during oogenesis and spermatogenesis and their persistence during early embryogenesis in the mouse. Genes Dev. 1, 1039-1046 https://doi.org/10.1101/gad.1.10.1039
  8. Santos, F., Hendrich, B., Reik, W., and Dean, W. (2002). Dynamic reprogramming of DNA methylation in the early mouse embryo. Dev. Biol. 241, 172-182 https://doi.org/10.1006/dbio.2001.0501
  9. Ealy, A.D., and Hansen, P.J. (1994). Induced thermotolerance during early development of murine and bovine embryos. J. Cell. Physiol. 160, 463-468 https://doi.org/10.1002/jcp.1041600309
  10. Gwazdauskas, F.C., McCaffrey, C., McEvoy, T.G., and Sreenan, J.M. (1992). In vitro preimplantation mouse embryo development with incubation temperatures of 37 and 39 degrees C. J. Assist Reprod. Genet 9, 149-154 https://doi.org/10.1007/BF01203755
  11. Lau, M.M., Stewart, C.E., Liu, Z., Bhatt, H., Rotwein, P., and Stewart, C.L. (1994). Loss of the imprinted IGF2/cationindependent mannose 6-phosphate receptor results in fetal overgrowth and perinatal lethality. Genes Dev. 8, 2953-2963 https://doi.org/10.1101/gad.8.24.2953
  12. Ozawa, M., Hirabayashi, M., and Kanai, Y. (2002). Developmental competence and oxidative state of mouse zygotes heat-stressed maternally or in vitro. Reproduction 124, 683-89 https://doi.org/10.1530/rep.0.1240683
  13. Surani, M.A. (1998) Imprinting and the initiation of gene silencing in the germ line. Cell 93, 309-312 https://doi.org/10.1016/S0092-8674(00)81156-3
  14. Doherty, A.S., Mann, M.R., Tremblay, K.D., Bartolomei, M.S., and Schultz, R.M. (2000). Differential effects of culture on imprinted H19 expression in the preimplantation mouse embryo. Biol. Reprod. 62, 1526-1535 https://doi.org/10.1095/biolreprod62.6.1526
  15. Khosla, S., Dean, W., Brown, D., Reik, W., and Feil, R. (2001). Culture of preimplantation mouse embryos affects fetal development and the expression of imprinted genes. Biol. Reprod. 64, 918-926 https://doi.org/10.1095/biolreprod64.3.918
  16. Aroyo, A., Yavin, S., Arav, A., and Roth, Z. (2007). Maternal hyperthermia disrupts developmental competence of follicleenclosed oocytes: in vivo and ex vivo studies in mice. Theriogenology 67, 1013-1021 https://doi.org/10.1016/j.theriogenology.2006.12.001
  17. Howlett, S.K., and Reik, W. (1991). Methylation levels of maternal and paternal genomes during preimplantation development. Development 113, 119-127
  18. Arechiga, C.F., Ealy, A.D., and Hansen, P.J. (1995). Evidence that glutathione is involved in thermotolerance of preimplantation murine embryos. Biol. Reprod. 52, 1296-1301 https://doi.org/10.1095/biolreprod52.6.1296
  19. Lefebvre, L., Viville, S., Barton, S.C., Ishino, F., Keverne, E.B., and Surani, M.A. (1998). Abnormal maternal behaviour and growth retardation associated with loss of the imprinted gene Mest. Nat. Genet. 20, 163-169 https://doi.org/10.1038/2464
  20. Li, L., Keverne, E.B., Aparicio, S.A., Ishino, F., Barton, S.C., and Surani, M.A. (1999). Regulation of maternal behavior and offspring growth by paternally expressed Peg3. Science 284, 330-333 https://doi.org/10.1126/science.284.5412.330
  21. Young, L.E., Fernandes, K., McEvoy, T.G., Butterwith, S.C., Gutierrez, C.G., Carolan, C., Broadbent, P.J., Robinson, J.J., Wilmut, I., and Sinclair, K.D. (2001). Epigenetic change in IGF2R is associated with fetal overgrowth after sheep embryo culture. Nat. Genet. 27, 153-154 https://doi.org/10.1038/84769
  22. Chen, T., Jiang, Y., Zhang, Y.L., Liu, J.H., Hou, Y., Schatten, H., Chen, D.Y., and Sun, Q.Y. (2005). DNA hypomethylation of individual sequences in aborted cloned bovine fetuses. Front Biosci. 10, 3002-3008 https://doi.org/10.2741/1756
  23. Tucker, K.L., Beard, C., Dausmann, J., Jackson-Grusby, L., Laird, P.W., Lei, H., Li, E., and Jaenisch, R. (1996). Germline passage is required for establishment of methylation and expression patterns of imprinted but not of nonimprinted genes. Genes Dev. 10, 1008-1020 https://doi.org/10.1101/gad.10.8.1008
  24. Gwazdauskas, F.C., Thatcher, W.W., and Wilcox, C.J. (1973). Physiological, environmental, and hormonal factors at insemination which may affect conception. J. Dairy Sci. 56, 873-877 https://doi.org/10.3168/jds.S0022-0302(73)85270-1
  25. Tremblay, K.D., Duran, K.L., and Bartolomei, M.S. (1997). A 5' 2-kilobase-pair region of the imprinted mouse H19 gene exhibits exclusive paternal methylation throughout development. Mol. Cell. Biol. 17, 4322-4329 https://doi.org/10.1128/MCB.17.8.4322
  26. Edwards, J.L., and Hansen, P.J. (1997). Differential responses of bovine oocytes and preimplantation embryos to heat shock. Mol. Reprod. Dev. 46, 138-145 https://doi.org/10.1002/(SICI)1098-2795(199702)46:2<138::AID-MRD4>3.0.CO;2-R
  27. Ozawa, M., Matsuzuka, T., Hirabayashi, M., and Kanai, Y. (2004). Redox status of the oviduct and CDC2 activity in 2-cell stage embryos in heat-stressed mice. Biol. Reprod. 71, 291-296 https://doi.org/10.1095/biolreprod.103.022152
  28. Sato, A., Otsu, E., Negishi, H., Utsunomiya, T., and Arima, T. (2007). Aberrant DNA methylation of imprinted loci in superovulated oocytes. Hum. Reprod. 22, 26-35 https://doi.org/10.1093/humrep/del316
  29. Edwards, J.L., King, W.A., Kawarsky, S.J., and Ealy, A.D. (2001). Responsiveness of early embryos to environmental insults: potential protective roles of HSP70 and glutathione. Theriogenology 55, 209-223 https://doi.org/10.1016/S0093-691X(00)00455-6
  30. Rivera, R.M., and Hansen, P.J. (2001). Development of cultured bovine embryos after exposure to high temperatures in the physiological range. Reproduction 121, 107-115 https://doi.org/10.1530/rep.0.1210107
  31. Kim, S.H., Kang, Y.K., Koo, D.B., Kang, M.J., Moon, S.J., Lee, K.K., and Han, Y.M. (2004). Differential DNA methylation reprogramming of various repetitive sequences in mouse preimplantation embryos. Biochem. Biophys. Res. Commun. 324, 58-63 https://doi.org/10.1016/j.bbrc.2004.09.023