Disrupted imprinting status at the H19 differentially methylated region is associated with the resorbed embryo phenotype in rats
Shilpa Pathak A , Madhurima Saxena A , Ryan D'Souza A and N. H. Balasinor A BA Division of Neuroendocrinology, National Institute for Research in Reproductive Health, Indian Council of Medical Research, JM Road, Parel, Mumbai 400 012, India.
B Corresponding author. Email: balasinorn@nirrh.res.in
Reproduction, Fertility and Development 22(6) 939-948 https://doi.org/10.1071/RD09154
Submitted: 1 July 2009 Accepted: 20 January 2010 Published: 25 May 2010
Abstract
Igf2, an imprinted gene that is paternally expressed in embryos, encodes an embryonic growth factor. An important regulator of Igf2 expression is methylation of the H19 differentially methylated region (DMR). A significant association has been observed between sperm methylation status at the H19 DMR and post-implantation loss. In addition, tamoxifen treatment has been shown to increase post-implantation loss and reduce DNA methylation at the H19 DMR in rat spermatozoa. Because this DMR is a primary DMR transmitting epigenetic imprint information from the gametes to the embryo, the aim of the present study was to determine the imprinting status of H19 DMR in post-implantation normal and resorbed embryos (F1) and to compare it with the H19 DMR in the spermatozoa of the respective sires. Analysis of the H19 DMR revealed methylation errors in resorbed embryo that were also observed in their sires' spermatozoa in the control and tamoxifen-treated groups. Expression analysis of the reciprocally imprinted genes Igf2 and H19 showed significant downregulation of Igf2 protein without any effect on H19 transcript levels in the resorbed embryos. The results indicate an association between disrupted imprinting status at the H19 DMR in resorbed embryos and the spermatozoa from their respective sires regardless of treatment, implying a common mechanism of resorption. The results demonstrate transmission of methylation errors at the Igf2–H19 locus through the paternal germline to the subsequent generation, emphasising the role of paternal factors during embryogenesis.
Additional keywords: DNA methylation, resorption, tamoxifen.
Acknowledgements
The study (NIRRH/MS/41/2008) was supported by the Indian Council of Medical Research, New Delhi, India, under ‘Genomics’. The authors thank Dr A. Maitra, Mr C. Saravanan and Ms U. Nanda for technical assistance with DNA sequencing. The authors also acknowledge the assistance for animal experimentation of Mr H. G. Pawar, Mr S. Mandavkar and Mr D. Shelar. The financial assistance provided by the Council of Scientific and Industrial Research (CSIR), New Delhi, to Ms Shilpa Pathak for carrying out her doctoral study is sincerely acknowledged.
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