Expression of some X-chromosome linked genes has recently been shown to be altered in bovine somatic cell nuclear transfer (SCNT) derived embryos (Wrenzycki et al. 2002 Biol. Reprod. 66, 127), implying that the regulatory mechanisms of X-linked transcription are affected by embryo in vitro production (IVP) methods. We analyzed the transcriptional pattern of X-linked genes (BIRC4, GAB3, HPRT1, MECP2, RPS4X, SLC25A6, and XIST) in bovine in vitro fertilized (IVF) and SCNT male and female blastocysts to determine X-inactivation status and changes resulting from IVP. We collected pools of male (n = 5 pools) and female (n = 3 pools) IVF-derived blastocysts (Bousquet et al. 1999 Theriogenology 51, 59) and male (n = 5 pools) and female (n = 3 pools) SCNT-derived blastocysts (Mastromonaco et al. 2004 Reprod. Domest. Anim. 39, 462). Each pool consisted of five blastocysts. Embryos were washed in phosphate buffered saline (PBS) + 0.1% polyvinyl alcohol (PVA), collected, and stored at -80�C. Total RNA was extracted with an Absolutely RNA Microprep kit (Stratagene, La Jolla, CA, USA), DNase I treated, and precipitated with isopropanol and linear acrylamide (Ambion, Inc., Austin, TX, USA) as a carrier. Reverse transcription was performed with Oligo-dT (Invitrogen, Burlington, Ontario, Canada) and Superscript II RT (Invitrogen). Transcript quantification was performed by quantitative real-time PCR using SYBR Green I (LightCycler system, Roche, Diagnostics, Laval, Quebec, Canada). Data analysis was performed with SAS (SAS Institute, Inc., Cary, SC, USA) using a mixed-model factorial ANOVA and with results presented as estimates of the median, ratios of estimates, and 95% confidence intervals with � = 0.05. IVF-derived male and female blastocysts possessed similar levels of the transcripts analyzed, suggesting successful dosage compensation at this developmental stage for embryos fertilized in vitro. XIST was not detected in male IVF embryos. GAB3 was not detected in any of the female groups and, in addition, HPRT1 transcripts were not detected in SCNT derived female embryos. Male and female SCNT-derived blastocysts possessed marked differences in their transcript levels, with males showing statistically significantly higher levels of BIRC4 and RPS4X and females possessing higher levels of MECP2 and SLC25A6 transcripts although differences between the latter two were not statistically significant. XIST was detected in both male and female SCNT blastocysts. We conclude that dosage compensation between male and female IVF blastocysts is achieved at this developmental stage for the transcripts examined. However, this pattern was markedly changed in the SCNT group, affecting especially female SCNT blastocysts, suggesting that the regulatory mechanisms of X-inactivation and X-linked gene expression are substantially altered in SCNT embryos probably due to aberrant epigenetic patterns and faulty genome reprogramming. We are currently analyzing X-linked transcription in male and female in vivo-derived blastocysts in order to compare this group with IVP-derived embryos.
This work was funded by NSERC, CIHR, and CRC.
Tissue, cell type and stage-specific ectopic gene expression and RNAi induction in the Drosophila testis