Analysis of the expression of putatively imprinted genes in bovine peri-implantation embryos P.Y. Tveden-Nyborg a, * , N.I. Alexopoulos a,b , M.A. Cooney b , A.J. French c,d , R.T. Tecirlioglu c , M.K. Holland b , P.D. Thomsen a , N.T. D’Cruz b a Dep. of Basic Animal and Veterinary Sciences, Groennegaardsvej 7, DK-1870 Frederiksberg C, Denmark b Monash Institute of Medical Research, Monash University, Clayton, VIC, Australia 1 c Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, VIC, Australia d Stemagen, Corp., La Jolla, CA, USA Received 21 August 2007; received in revised form 24 April 2008; accepted 7 June 2008 Abstract The application of assisted reproductive technologies (ART) has been shown to induce changes in the methylation of the embryonic genome, leading to aberrant gene expression, including that of imprinted genes. Aberrant methylation and gene expression has been linked to the large offspring syndrome (LOS) in bovine embryos resulting in increased embryonic morbidity and mortality. In the bovine, limited numbers of imprinted genes have been studied and studies have primarily been restricted to pre- implantation stages. This study reports original data on the expression pattern of 8 putatively imprinted genes (Ata3, Dlk1, Gnas, Grb10, Magel2, Mest-1, Ndn and Sgce) in bovine peri-implantation embryos. Two embryonic developmental stages were examined, Day 14 and Day 21. The gene expression pattern of single embryos was recorded for in vivo, in vitro produced (IVP) and parthenogenetic embryos. The IVP embryos allow us to estimate the effect of in vitro procedures and the analysis of parthenogenetic embryos provides provisional information on maternal genomic imprinting. Among the 8 genes investigated, only Mest-1 showed differential expression in Day 21 parthenogenetic embryos compared to in vivo and IVP counterparts, indicating maternal imprinting of this gene. In addition, our expression analysis of single embryos revealed a more heterogeneous gene expression in IVP than in in vivo developed embryos, adding further to the hypothesis of transcriptional dysregulation induced by in vitro procedures, either by in vitro maturation, fertilization or culture. In conclusion, effects of genomic imprinting and of in vitro procedures for embryo production may influence the success of bovine embryo implantation. # 2008 Elsevier Inc. All rights reserved. Keywords: Gene expression; Mest-1; Peri-implantation; Uniparental; Bovine 1. Introduction Epigenetic mechanisms have been shown to criti- cally influence embryonic development through the control of gene expression and chromatin packaging. Of the different modes of epigenetic regulation of DNA transcription, imprinted genes comprise a specific subgroup. This subgroup is characterized by a mono- allelic expression depending on parental origin, with no change to the genetic code [1–3]. During pre- implantation embryonic development imprinted genes escape the global demethylation of the parental genomes and are able to display their parental specific monoallelic expression [4]. The conventional imprinted www.theriojournal.com Available online at www.sciencedirect.com Theriogenology 70 (2008) 1119–1128 * Corresponding author. Tel.: +45 353 33167. E-mail address: ptn@life.ku.dk (P.Y. Tveden-Nyborg). 1 In cooperation with the Co-operative Research Centre for Inno- vative Dairy Products. 0093-691X/$ – see front matter # 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2008.06.033