236 SPATIAL GENE EXPRESSION PATTERNS OF Dnmt1, Dnmt3a, AND Hdac2 IN PREIMPLANTATION EMBRYOS

Abstract

In mammals, DNA methylation and the modification of histones account for the major epigenetic alterations. Usually DNA methylation is associated with transcriptional silencing, which is catalyzed by two important classes of DNA methyltransferases. DNA methyltransferase 1 (Dnmt1), a maintenance enzyme, methylates hemi-methylated DNA after DNA replication. Dnmt3a and Dnmt3b are required for de novo methylation in vivo and for establishing new DNA methylation marks during development. In addition to DNA methylation, post-translational modifications of the amino termini of core histones are thought to affect the expression or repression of transcription. Histone deacetylation catalyzsed by histone deacetylases (Hdac) often results in transcriptional repression. Perturbated epigenetic reprogramming of the genome is a likely cause of developmental abnormalities and epigenetic diseases introduced by assisted reproduction technologies. The objective of the present study was to determine the relative abundance of Dnmt1, Dnmt3a, and Hdac2 transcripts in ICM and TE cells of pre-implantation bovine embryos of different origin. Embryos were generated with standard protocols of in vitro production (IVP) and parthenogenetic activation using SOF medium supplemented with BSA (SOF d7/d8, Parth d7/d8) or TCM medium supplemented with estrus cow serum (TCM d7/d8; Wrenzycki et al. 2001 Hum. Reprod. 16, 893–901; Wrenzycki et al. 2002 Biol. Reprod. 66, 127–134). Expanded blastocysts were collected at Days 7 and Day 8 (d7/d8) of culture (Day 0 = IVF). In vivo-generated blastocysts collected from superovulated animals were included as controls (Wrenzycki et al. 1996 J. Reprod. Fert. 108, 17–24). A highly sensitive RT-PCR assay (Wrenzycki et al. 2003 Biol. Reprod. 68, 2073–2080) was used to determine the relative abundance (RA) of gene transcripts in isolated ICM and TE cells. The allocation of ICM and TE cells was analyzed using a differential staining technique (Eckert and Niemann 1998 Mol. Hum. Reprod. 4, 957–965) to calculate the RA of each transcript on a per cell basis. RT-PCR assays were repeated at least six times. Significant differences in the RA of Dnmt1 (In vivo, TCM d7, Parth d7/d8, SOF d8), Dnmt3a (In vivo, SOF d7), and Hdac2 (TCM d7) transcripts were detected between ICM and TE cells. No differences were detected for all transcripts in ICM cells of embryos collected at either Day 7 or Day 8. In TE cells, the RA of Dnmt1 transcripts was significantly reduced or increased in embryos generated in SOF or TCM medium at Day 7, respectively, and the RA of Hdac2 transcripts was significantly higher in embryos generated in TCM medium at Day 8. These results suggest that in vitro culture alters the spatial expression pattern of genes related to epigenetic modifications in the cell lineages critical for a normal embryonic and fetal development.

This work was supported by the Deutsche Forschungsgemeinschaft (DFG Ni 256/18-1).