Expression profiles of the pluripotency marker gene POU5F1 and validation of reference genes in rabbit oocytes and preimplantation stage embryos.

BACKGROUND: The surge in the number of gene expression studies and tendencies to increase the quality of analysis have necessitated the identification of stable reference genes. Although rabbits are classical experimental model animals, stable reference genes have not been identified for normalization. The aims of this study were to compare the expression profiles of the widely used reference genes in rabbit oocytes and preimplantation stage embryos, and to select and validate stable ones to use as reference. RESULTS: Quantitative real time PCR method was used to evaluate 13 commonly used references (Actb, Gapdh, Hprt1, H2afz, Ubc, Ppia, Eef1e1, Polr2a, Tbp, G6pdx, B2m, Pgk1, and Ywhaz) and POU5F1 (Oct4) genes. Expressions of these genes were examined in multiple individual embryos of seven different preimplantation developmental stages and embryo types (in vivo and in vitro). Initial analysis identified three genes (Ubc, Tbp, and B2m) close to the detection limit with irregular expression between the different stages. As variability impedes the selection of stable genes, these were excluded from further analysis. The expression levels of the remaining ten genes, varied according to developmental stage and embryo types. These genes were ranked using the geNorm software and finally the three most stable references (H2afz, Hprt1, and Ywhaz) were selected. Normalization factor was calculated (from the geometric averages of the three selected genes) and used to normalize the expressions of POU5F1 gene. The results showed the expected expression patterns of the POU5F1 during development. CONCLUSION: Compared to the earlier studies with similar objectives, the comparison of large number of genes, the use of multiple individual embryos as compared to pools, and simultaneous analyses of in vitro and in vivo derived embryo samples were unique approaches in our study. Based on quantification, pattern and geNorm analyses, we found the three genes (H2afz, Hprt1, and Ywhaz) to be the most stable across developmental stages and embryo types, and the geometric averages of these genes can be used for appropriate normalization.

Quantitative evaluation and selection of reference genes in mouse oocytes and embryos cultured in vivo and in vitro.

BACKGROUND: Real-time PCR is an efficient tool to measure transcripts and provide valuable quantitative information on gene expression of preimplantation stage embryos. Finding valid reference genes for normalization is essential to interpret the real-time PCR results accurately, and understand the biological dynamics during early development. The use of reference genes also known as housekeeping genes is the most widely applied approach. However, the different genes are not systematically compared, and as a result there is no uniformity between studies in selecting the reference gene. The goals of this study were to compare a wide selection of the most commonly used housekeeping genes in mouse oocytes and preimplantation stage embryos produced under different culture conditions, and select the best stable genes for normalization of gene expression data. RESULTS: Quantitative real time PCR method was used to evaluate 12 commonly used housekeeping genes (Actb, Gapdh, H2afz, Hprt, Ppia, Ubc, Eef1e1, Tubb4, Hist2h2aa1, Tbp, Bmp7, Polr2a) in multiple individual embryos representing six different developmental stages. The results were analysed, and stable genes were selected using the geNorm software. The expression pattern was almost similar despite differences in the culture system; however, the transcript levels were affected by culture conditions. The genes have showed various stabilities, and have been ranked accordingly. CONCLUSION: Compared to earlier studies with similar objectives, we used a unique approach in analysing larger number of genes, comparing embryo samples derived in vivo or in vitro, analysing the expression in the early and late maternal to zygote transition periods separately, and using multiple individual embryos. Based on detailed quantification, pattern analyses and using the geNorm application, we found Ppia, H2afz and Hprt1 genes to be the most stable across the different stages and culture conditions, while Actb, the classical housekeeping gene, showed the least stability. We recommend the use of the geometric averages of those three genes for normalization in mouse preimplantation-stage gene expression studies.

Gene expression profiles and in vitro development following vitrification of pronuclear and 8-cell stage mouse embryos.

The analysis of differences in gene expression, responding to cryopreservation may explain some of the observed differences in further development of the preimplantation stage embryos. The aim of this study was to create a link, for the first time, between morphological/developmental observations and gene activity changes following cryopreservation of embryos. Efficiency of two vitrification methods, solid surface and in-straw vitrifications for pronuclear-stage mouse zygotes and 8-cell stage mouse embryos was compared based on morphological survival, blastocyst formation, and changes in embryonic gene expression. Both stages of embryos were vitrified by SSV using 35% ethylene glycol (EG) for vitrification solution (VS) and in-straw vitrification using 40% EG for VS. No significant differences were found between immediate survival rates of embryos vitrified by SSV and in-straw vitrification in both stages. Blastocyst rates were significantly higher with SSV and not significantly different from that of control. These results showed that SSV was more efficient than in-straw vitrification. Treatment with cytochalasin-b did not improve cryosurvival during SSV. The quantification of selected gene transcripts from single embryo (6 embryos/treatment group) were carried out by quantitative real-time RT-PCR. It was performed by adding 1/8 of each embryo cDNA to the PCR mix containing the specific primers to amplify housekeeping gene (beta-actin), heat shock protein gene (Hsp70), genes related to oxidative stress (MnSOD and CuSOD), cold stress (CirpB, Rbm3), and cell-cycle arrest (Trp53). We found upregulation of all six stress-related genes at 3 hr post-warming in pronuclear stage embryos. Expression of these genes showed much higher level (2-33-fold) in in-straw vitrification than in in vitro control embryos. In SSV-treated embryos we could detect only slight changes (0.3-2-fold). At 10 hr post-warming, all genes were downregulated in embryos vitrified by in-straw method. In SSV-treated group expression of Hsp70 showed slight increase and Trp53 showed decrease. In contrast to pronuclear stage, there was no clear pattern of gene expression changes after vitrification in 8-cell stage embryos. Several genes were upregulated both at 3 and 10 hr post-warming. Moreover, we found upregulation of beta-actin gene which we expected to use as a reference gene in in-straw treated embryos in both 3 and 10 hr post-warming, while in pronuclear stage embryos and in SSV treatment there was no effect on beta-actin expression level. There was no difference in gene expression between blastocysts developed from fresh or vitrified embryos. In conclusion, the real-time RT-PCR method from single embryo opened new opportunities for the understranding of molecular events following cryopreservation. The upregulation of stress-related genes at 3 hr post-warming in pronuclear stage embryos might have been an early indicator of reduced viability following in-straw vitrification in good correlation with the developmental data to blastocyst stage.

Comparison of real-time polymerase chain reaction and end-point polymerase chain reaction for the analysis of gene expression in preimplantation embryos.

The aim of the present study was to compare real-time polymerase chain reaction (PCR) and end-point PCR with respect to their suitability for the analysis of gene expression in samples in which the number of cells is limited; for example, in studies of preimplantation embryonic development and to determine the variability of the real-time reverse transcription-PCR assay. The sensitivity, dynamic range and precision of both PCR systems were compared using a single mouse liver cDNA standard. The real-time system was 100-fold more sensitive than the end-point system and had a dynamic range of more than four orders of magnitude. The linear range for end-point PCR extended for two orders of magnitude using a fixed end-point of 31 cycles. The percentage standard error of the mean based on 30 replicates was 0.14% of the threshold cycle (Ct) value for the real-time system and 6.8% for the end-point fluorescence intensity. The coefficients of variation (CV) for reverse transcription combined with real-time analysis and the complete gene expression protocol consisting of mRNA isolation, reverse transcription and real-time PCR analysis were 0.6% and 1.4% of the Ct values, respectively. The present paper details, for the first time, measurement of the biological variation of individual mammalian oocytes. The CV was 1.8% of the Ct value for expression analysis of six bovine oocytes. The results are discussed in relation to the analysis of gene expression in preimplantation embryo development.