234 IMPRINTING STATUS OF DEVELOPMENTALLY IMPORTANT GENES IN BOVINE PREIMPLANTATION EMBRYOS

Abstract

Several imprinted genes have been identified in different species such as mouse and man but in cattle imprinting has only been confirmed for the Insulin-like growth factor 2 receptor gene (Igf2r). DNA methylation is one of the most common mechanisms of imprinting. Imprinting is correlated with the methylation of normally unmethylated CpG islands, and aberrations in methylation are thought to be involved in the Large Offspring Syndrome (LOS) which is frequently observed in offspring derived from in vitro-produced and/or cloned embryos. The imprinting of the bovine Insulin-like growth factor 2 gene (Igf2), the Igf2r gene and the Mammalian achaete scute homologue 2 gene (Mash2) was analyzed in bovine preimplantation blastocysts cultured in SOF + BSA. mRNA levels of the Igf2r gene and the Mash2 gene in in vitro-produced (IVP) and parthenogenetic expanded single blastocysts were analyzed by semi-quantitative RT-PCR (Wrenzycki C et al., 2001 Biol. Reprod. 65, 309–317). Genes expressed exclusively from the maternal allele (i.e. paternally methylated) should be represented by a higher relative abundance of transcriptional products in parthenogenetic embryos whereas paternally expressed genes (i.e. maternally methylated) should be correlated with a higher gene expression in IVP embryos carrying one paternal and one maternal allele. For determination of methylation patterns by bisulfite sequencing, sequences of several DNA fragments from the bovine Igf2 gene were identified in samples from bovine uterus and kidney. The primer pairs were generated from the ovine and bovine Igf2 sequences available in the Genebank database (accession number U00664/X53553). DNA fragments identified were from the 5′ untranslated region and the 3′ translated region of the bovine Igf2 gene. All fragments consist of a high number of CG dinucleotides, and computer analysis using the CpGwin program (Anbazhagan R et al., 2001 BioTechniques 30, 110–114) revealed CpG islands within these fragments. Relative abundances of transcriptional products were statistically analyzed using the SigmaStat 2.0 (Jandel Scientific, San Rafael, CA, USA) software package. After testing for normality, an ANOVA followed by multiple pairwise comparisons using the Tukey test was employed. Results from the semi-quantitative RT-PCR analyses revealed no difference (P > 0.05) in gene expression pattern of the Igf2r gene, suggesting that the Igf2r gene is biallelically expressed during bovine preimplantation development. In contrast, there was a significant difference (P < 0.05) in the relative abundance of mRNA of the Mash2 gene, with a lower relative abundance of transcriptional products in parthenogenetic expanded blastocysts. Thus, in cattle as reported in mice (Guillemot F et al., 1995 Nat. Genet. 9, 235–242), the paternal allele of the Mash2 gene seems to participate in the expression of the gene prior to implantation.