In vitro culture conditions or the SCNT process affect changes in X-linked gene expression and methylation in preimplantation embryos, which in turn can lead to long-term effects [15,19,33]. Our results show that both IVF and cloned embryos exhibit aberrant expression, with either up- or downregulation, for several genes, although the average levels of all X-linked gene mRNAs that were tested showed sex-specific expression. Also, somewhat distinctive patterns of gene expression were observed get Teriparatide between IVF and cloned embryos, as well as between the sexes. Among these genes, BEX1 exhibited variable gene expression in only a small subset of IVF embryos, while others fell within the normal range. This was in contrast to observations in cloned blastocysts in which variable gene expression was observed in a large subset of individuals, suggesting that dysregulated BEX1 may be largely due to incomplete reprogramming following the SCNT process, rather than in vitro culture. Previous studies on haploid mouse parthenotes have suggested that upregulated Bex1 expression may affectX-Linked Gene Transcripts in Pig BlastocystsFigure 7. X-linked gene transcription patterns of pFF cell lines. Asterisks indicate significant difference between the different cell lines (P,0.01). doi:10.1371/journal.pone.0051398.gcommitment to the trophectoderm lineage, which in turn could arrest development [34,35]. Thus, determining if the observed aberrant expression of BEX1 can influence further embryonic development in cloned porcine embryos will be interesting. The data showed relatively stable expression patterns for G6PD and HPRT1 in IVF and cloned blastocysts. Levels of G6PD and HPRT transcripts were also higher in JWH133 site female blastocysts than in males, but only HPRT mRNA levels in IVF and cloned embryos were comparable to those in in vivo embryos. G6PD showed upregulated expression in IVF embryos of both sexes compared with their in vivo counterparts. This observation is consistent with a previous report showing increased expression of this gene in bovine IVF embryos [14]. Repressed Pgk1 expression in cloned mouse embryos was found by Fukuda et al. [36], which is consistent with our results. However, we also found that PGK1 was consistently downregulated in IVF embryos, indicating that aberrant expression of this gene may not be solely due to the SCNT process but may be attributable to in vitro cultures. Such downregulation of the glycolysis-related PGK1 gene in both types of in vitro embryos may be due to our culture conditions, when glucose in the PZM3 media was depleted. The reported onset of compensation for the Hprt and Pgk1 gene dosages between XX and XY mouse embryos before theblastocyst stage [25] is in contrast to our result of an apparent sex-biased difference in porcine blastocysts for the expression of most of the tested X-linked genes. These results indicate that differences in timing to acquire compensation for X dosages may exist across mammalian species. Upregulated XIST expression was observed in both IVF and cloned embryos. Higher Xist expression in cloned embryos provides evidence that ectopic Xist expression from the Xa leads to abnormal XCI and is responsible for genome-wide downregulation [19]. Despite upregulated XIST expression in IVF and cloned embryos, we could not find repressed patterns for the other X-linked genes, except for PGK1, which may respond to environmental factors like the in vitro culture or manipulations. In the mouse, Xist is in.In vitro culture conditions or the SCNT process affect changes in X-linked gene expression and methylation in preimplantation embryos, which in turn can lead to long-term effects [15,19,33]. Our results show that both IVF and cloned embryos exhibit aberrant expression, with either up- or downregulation, for several genes, although the average levels of all X-linked gene mRNAs that were tested showed sex-specific expression. Also, somewhat distinctive patterns of gene expression were observed between IVF and cloned embryos, as well as between the sexes. Among these genes, BEX1 exhibited variable gene expression in only a small subset of IVF embryos, while others fell within the normal range. This was in contrast to observations in cloned blastocysts in which variable gene expression was observed in a large subset of individuals, suggesting that dysregulated BEX1 may be largely due to incomplete reprogramming following the SCNT process, rather than in vitro culture. Previous studies on haploid mouse parthenotes have suggested that upregulated Bex1 expression may affectX-Linked Gene Transcripts in Pig BlastocystsFigure 7. X-linked gene transcription patterns of pFF cell lines. Asterisks indicate significant difference between the different cell lines (P,0.01). doi:10.1371/journal.pone.0051398.gcommitment to the trophectoderm lineage, which in turn could arrest development [34,35]. Thus, determining if the observed aberrant expression of BEX1 can influence further embryonic development in cloned porcine embryos will be interesting. The data showed relatively stable expression patterns for G6PD and HPRT1 in IVF and cloned blastocysts. Levels of G6PD and HPRT transcripts were also higher in female blastocysts than in males, but only HPRT mRNA levels in IVF and cloned embryos were comparable to those in in vivo embryos. G6PD showed upregulated expression in IVF embryos of both sexes compared with their in vivo counterparts. This observation is consistent with a previous report showing increased expression of this gene in bovine IVF embryos [14]. Repressed Pgk1 expression in cloned mouse embryos was found by Fukuda et al. [36], which is consistent with our results. However, we also found that PGK1 was consistently downregulated in IVF embryos, indicating that aberrant expression of this gene may not be solely due to the SCNT process but may be attributable to in vitro cultures. Such downregulation of the glycolysis-related PGK1 gene in both types of in vitro embryos may be due to our culture conditions, when glucose in the PZM3 media was depleted. The reported onset of compensation for the Hprt and Pgk1 gene dosages between XX and XY mouse embryos before theblastocyst stage [25] is in contrast to our result of an apparent sex-biased difference in porcine blastocysts for the expression of most of the tested X-linked genes. These results indicate that differences in timing to acquire compensation for X dosages may exist across mammalian species. Upregulated XIST expression was observed in both IVF and cloned embryos. Higher Xist expression in cloned embryos provides evidence that ectopic Xist expression from the Xa leads to abnormal XCI and is responsible for genome-wide downregulation [19]. Despite upregulated XIST expression in IVF and cloned embryos, we could not find repressed patterns for the other X-linked genes, except for PGK1, which may respond to environmental factors like the in vitro culture or manipulations. In the mouse, Xist is in.
