340 EPIGENETIC ANALYSIS OF DEVELOPMENTALLY IMPORTANT GENES IN BOVINE OOCYTES OF DIFFERENT ORIGINS

Abstract

A critical step in assisted reproductive technologies (ART) is the IVM of oocytes. The quality of the oocyte is crucial for successful fertilization and subsequent embryo development. Studies in bovine ART, and epidemiological studies in children from ART, reveal a degree of abnormal development thought to be primarily caused by aberrant DNA methylation patterns in imprinted and non-imprinted genes. Due to the inherent similarities in bovine and human preimplantation embryonic development, bovine oocyte and embryo development is increasingly being used as a model for human development. The goal of this project is to investigate the effects of specific IVM conditions on the DNA methylation profile of bovine oocytes and early embryos and to determine the relationship between DNA methylation and mRNA expression for selected developmentally important genes (PEG3, IGF2R, SNRPN, GDF9, Glut8, PRDX1, Dnmt1A and Dnmt3a/b). Two different IVM systems have been established, the first is the routinely used TCM-199 medium at 39°C, 5% CO₂ in air, the second uses synthetic oviduct fluid medium (SOF) as the basic medium with minor modifications (10 mM glucose, 1 mM glutamine) at 39°C, 5% CO₂ and reduced oxygen tension (5%). Oocytes for IVM were retrieved by slicing of ovaries collected from local slaughterhouses. Successful maturation was confirmed by extrusion of the first polar body for 78% (TCM) and 72% (SOF) of the oocytes, respectively. Results of quantitative real-time PCR experiments indicate that transcripts of the maternally imprinted gene PEG3 and the paternally imprinted gene IGF2R as well as transcripts of other developmentally important genes (GDF9, PRDX1 and Glut8) are present in immature and IVM oocytes and are applicable targets for further expression and methylation studies. Relative quantification of mRNA transcripts normalised to an external standard revealed that, while expression of the maternally imprinted PEG3 gene is lower in all groups of oocytes (immature: 9.8, matured in TCM: 3.3, and in SOF: 6.4), compared to the paternally imprinted IGF2R (immature: 29.3, TCM: 14.2, SOF: 19.2), no significant difference between the both groups of IVM oocytes could be detected. However, determination of specific effects of IVM will require more replicates and the comparison with IVM oocytes. This is currently underway and together with the ongoing methylation analysis based on the novel identification of differentially methylated regions in bovine imprinted genes, this study will provide deeper insight into the epigenetic profile of bovine oocytes as a potential causative mechanism implicated in the increased incidence of developmental abnormalities in human and bovine ART.

Funded by DFG Research Unit Germ Cell Potential.