P-82: Effect of SCNT Steps on Relative mRNA Abundances of Sheep Oocytes

Background: The oocyte is a unique cell committed to reprogram fertilizing sperm and to support early stages of embryonic development until the species-specific stage of zygote genome activation that occurs around the second to third cell cycle in sheep embryos. In this sense, considering the huge list of oocyte transcripts, we selected some candidates genes based on their roles of regulating different cell functions. Materials and Methods: The effect of zona removal and two main steps of SCNT (enucleation and nuclear transfer) on relative abundances of eight developmentally important genes (CCNB1, POU5F1, PAP, CX43, GMNN, NPM2, ATP1A1, and HSP90) and two housekeeping genes (GAPDH and ACTB) were studied and compared with the transcript profile of zona-intact oocytes. For this purpose, 30 minutes after zona removal, enucleation, and nuclear transfer, oocytes were used for quantitative realtime PCR. Results: For all genes assessed, neither zona removal nor enucleation significantly affected the relative abundances of transcripts, except for RNA abundance of CCNB1, which was significantly reduced following enucleation compared to zona-intact oocytes. However,transcript abundances of CCNB1, POU5F1, NPM2, GMMN, and CX43 were all significantly reduced after electrofusion of enucleated oocytes with the nuclei donor cells. Unlike other genes, the reduction in abundances of PAP, ATP1A1, and HSP90 was not affected by the SCNT steps. Conclusion: The RNA contents that were just affected by the reconstruction procedure were NPM2, POU5F1, CX43, and GMNN, which are involved in pluripotency, gap junction communication and regulation mechanisms, DNA replication, and reprogramming during early embryogenesis, respectively. These results suggested that SCNT steps have determining effects on oocyte transcripts of the reconstituted oocytes compared to intact counterparts.