84 GENE EXPRESSION DURING BLASTOCYST-TO-ELONGATED STAGE IN BOVINE EMBRYOS DERIVED FROM SOMATIC CELL NUCLEAR TRANSFER

Abstract

Epigenetic modification is an important factor in the development of embryos and the production of normal offspring derived from somatic cell nuclear transfer (NTSC). Several investigators have reported aberrant gene transcription in bovine NTSC embryos at the blastocyst (BC) stage. The objectives of this study were to evaluate the gene expression in NTSC embryos, which had developed to the elongated (EL) stage, and clarify differential levels of gene transcription in the embryo disc (ED) and trophectoderm (TE) of EL embryos. Five specific mRNAs [octamer-binding transcription factor (OCT-4), interferon-τ (IFN-τ), fibroblast growth factor receptor 2 (FGF-R2), and fibroblast growth factors 2 and 4 (FGF-2 and FGF-4)] were selected. Bovine BC embryos were obtained from NTSC using calf fibroblast cells or the uterus of donor cows after AI (Vivo). Some BC embryos were transferred to recipient cows at Day 7 (Day 0 = estrus), and then EL embryos were collected by uterine flushing at Day 16. Total RNA in single BC, ED, and TE were reverse-transcribed for PCR. Quantification of mRNA abundance was performed by real-time PCR. The expression of each mRNA was normalized to the abundance of GAPDH. A total of 15 (BC) and 7 (ED and TE) samples were used in each group to analyze the gene expression. Data on mRNA expression levels were analyzed using a Kruskal–Wallis test followed by multiple pair-wise comparisons using the Scheffe method. Most embryos (87–100%) gave positive signals of OCT-4, IFN-τ, and FGF-R2, regardless of the origin and stage of the embryos. Transcript signals of FGF-4 in BC embryos derived from Vivo (100%) and NTSC (70%) were detected with higher frequencies. At the EL stage, the FGF-4 signal was detected in only ED. The transcript of FGF-2 was detected with lower frequencies (20–27%) in BC embryos, but was consistently (71–86%) detected in ED of both groups. The relative abundance of OCT-4 expression in NTSC was higher (P < 0.05) than in Vivo embryos at the BC stage. In contrast, the transcript of FGF-4 at the BC stage was lower (P < 0.01) in NTSC than in Vivo embryos. Transcript levels of IFN-Ä, FGF-R2, and FGF-2 were not significantly different in both groups at the BC stage. The amount of OCT-4, FGF-4, and FGF-2 transcripts in ED were significantly (P < 0.05) higher than in TE. Transcript levels of these genes did not differ between NTSC and Vivo embryos. FGF-R2 levels were not significantly different in origins and tissue of EL embryos. In Vivo embryos, the IFN-τ level of TE was significantly (P < 0.05) higher than in ED. However, the difference in the IFN-τ transcription was not observed between ED and TE in NTSC embryos. The results of an analysis of mRNA transcripts at 2 different stages of development demonstrate that bovine NTSC embryos at the BC stage show deviations in expression patterns with respect to several genes which have important roles in cell differentiation, implantation, and embryo development, but these expressions, except for IFN-τ, were modified to a normal level according to the embryo development and differentiation.