Abstract
We examined the allelic expression and positioning of two pluripotency‐associated genes, OCT4 and SOX2, and two housekeeping genes, ACTB and TUBA, in 4‐ and 8‐cell porcine embryos utilizing RNA and DNA fluorescence in situ hybridization (FISH) in single blastomeres. The proportion of blastomeres expressing SOX2 bi‐allelically increased from 45% at the 4‐cell stage to 60% at the 8‐cell stage. Moreover, in 8‐cell embryos, SOX2 was expressed bi‐allelically in significantly more blastomeres than was the case for OCT4, and this was associated with a tendency for SOX2 alleles to move toward the nuclear interior during 4‐ to 8‐cell transition. However, the radial location of OCT4 alleles did not change significantly during this transition. The locations of active and inactive alleles based on DNA and RNA FISH signals were also calculated. Inactive OCT4 alleles were located in very close proximity to the nuclear membrane, whereas active OCT4 alleles were more centrally disposed in the nucleus. Nevertheless, the nuclear location of active and inactive SOX2 alleles did not change in either 4‐ or 8‐cell blastomeres. Our RNA and DNA FISH data provide novel information on the allelic expression patterns and positioning of pluripotency‐associated genes, OCT4 and SOX2, during embryonic genome activation in pigs.
Highlights
DNA is contained in the nucleus with heterochromatin concentrated at the periphery and around the nucleolus, and gene-rich regions are preferentially located in the interior of the nucleus (Cremer & Cremer, 2006; Gilbert, Gilchrist, & Bickmore, 2004; Misteli, 2007)
We examined the allelic expression pattern and nuclear location of two pluripotency-associated genes, OCT4 and SOX2, during embryonic genome activation in pigs
The location of these genes in the nucleus was determined in terms of the radial distance from the gene alleles to the nuclear membrane
Summary
DNA is contained in the nucleus with heterochromatin concentrated at the periphery and around the nucleolus, and gene-rich regions are preferentially located in the interior of the nucleus (Cremer & Cremer, 2006; Gilbert, Gilchrist, & Bickmore, 2004; Misteli, 2007). OCT4 shifts its localization from the interior to the surface of its chromosome territory in lymphoblastoid cells (Wiblin, 2005) These observations suggest that a large number of genes likely undergo repositioning during early embryonic development, especially during genome activation when the expression of many genes is significantly up- or downregulated. Maintenance of pluripotency in stem cells or early embryos requires tight regulation of key pluripotency-associated genes, and one possible mechanism for this may be mono-allelic expression (Miyanari & Torres-Padilla, 2012). Research on mouse embryos has suggested that the transcriptional activity of Nanog is subject to mono-allelic bursting and that this is crucial for maintenance of pluripotency, embryonic development, and reprogramming (Miyanari & Torres-Padilla, 2012). We examined the allelic expression pattern and repositioning of the pluripotency-associated genes, SOX2 and OCT4, in 4- and 8-cell embryos during embryonic genome activation in pigs
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