A comparative analysis of mRNA expression patterns between embryos produced under different in vitro and in vivo culture systems allows the isolation of genes associated with embryo quality and investigation of the effect of culture environment on the embryonic gene expression. In this study, expression analysis of four known (PSCD2, TCF7L2, NADH-subunit and PAIP1) genes and one novel transcript, derived from differential display PCR, was performed in in vitro (Ponsuksili et al., 2002, Theriogenology 57, 1611–1624) or in vivo- (Moesslacher et al., 2001 Reprod. Dom. Anim. 32, 37) produced bovine 2-, 4-, 8-, 16-cell, morula and blastocyst stage embryos using real time PCR technology. Poly(A) RNA was isolated from four separate individual embryos from each developmental stage and embryo group (in vitro or in vivo) using Dynabeads mRNA kit (Dynal, Oslo, Norway). After reverse transcription, quantitative PCR was performed with sequence specific primers in an ABI PRISM® 7000 Sequence Detection System instrument (Applied Biosystems, Foster City, CA, USA) using SYBR® Green as a double-strand DNA-specific fluorescent dye. Standard curves were generated for target and endogenous genes using serial dilutions of plasmid DNA. Final quantification was done using the relative standard curve method, and results were reported as relative expression or n-fold difference to the calibrator cDNA (i.e., the blastocyst stage) after normalization with the endogenous control (Histone2a). Data were analyzed using SAS version 8.0 (SAS Institute Inc., NC, USA) software package. Analysis of variance was performed with the main effects being the developmental stage and embryo source (in vitro or in vivo) and their interactions followed by multiple pairwise comparisons using Tukey’s test. No significant difference was observed in the relative abundance of the PSCD2 gene between the two embryo groups. However, its expression was higher (20-fold) (P<0.05) at the 8-cell stage than the other developmental stages among in vitro embryos. Higher expression (P<0.05) of NADH-subunit mRNA was detected in vivo than in vitro at the 2-cell stage of development. The TCF7L2 mRNA was expressed in the in vitro embryos but not in the in vivo ones. PAIP1 mRNA was higher (P<0.05) in in vitro (1500-fold) than in the in vivo embryos (500-fold) at the 2-cell developmental stage compared to the calibrator. The novel transcript was also detected at higher level (P<0.05) in the in vitro than in the in vivo embryos at the 2-cell stage of development. However, the PAIP1 and the novel transcript showed no significant difference in their expression between the two embryo groups beyond the 2-cell developmental stage. Both PAIP1 and the novel transcript were detected only up to 8-cell stage in both embryo groups, suggesting their maternal origin. In conclusion, the variations in the expression of studied genes between in vitro and in vivo may reflect the effect of the two culture systems on the transcriptional activity of early embryos.
Development of a RP-HPLC Method for Separating and Quantifying Muscimol in Different Developmental Stages of the Fungus Amanita muscaria
