| Autor: |
Ledgard, Anita M., Lee, Rita S-F., Couldrey, Christine, Peterson, A. J. |
| Zdroj: |
Biology of Reproduction; May 2008, Vol. 78 Issue: 1, Number 1 Supplement 1 p292-292, 1p |
| Abstrakt: |
Differential gene expression screening (using a macroarray of 192 candidate genes) was carried out on placental tissues obtained from Day 50 bovine AI and somatic cell nuclear transfer (SCNT) clone conceptuses. Dickkopf-1 (Dkk-1), a secreted glycoprotein that antagonizes the Wnt signaling pathway, was identified as down-regulated in both fetal and maternal SCNT cotyledon tissue compared to AI tissue. We examined the role of Dkk-1 in bovine placentation and whether its down-regulation in SCNT generated pregnancies was due to epigenetic factors such as DNA methylation differences. Real time PCR analysis confirmed the down-regulation of Dkk-1 mRNA at Day 50 of gestation in SCNT fetal cotyledons compared to AI and in SCNT maternal cotyledon tissue compared with those from AI pregnancies (2 fold less, P < 0.007). Analysis of Days 100 and 150 fetal cotyledons by slotblot showed that Dkk-1 was also down-regulated 2 fold in SCNT fetal cotyledons compared to AI at these stages (P < 0.007). Dkk-1 expression in trophoblast tissue was evident from as early as Day 18 and increased 10 fold by Day 24. Day 24-31 allantois and yolk sac tissue had very low levels of expression. Trophoblast tissue from SCNT embryos had 3 fold less Dkk-1 mRNA at Day 18 (P <0.001) and tended to have less at Day 26 than AI trophoblast. Western blotting showed protein levels of Dkk-1 in the fetal cotyledon tissue reflected the reduced gene expression in SCNT cotyledons. Placental problems contribute to failure in SCNT pregnancies and it has been postulated that this is due to improper epigenetic reprogramming during SCNT. Dkk-1 is a candidate for inappropriate reprogramming of DNA methylation sites as it has been shown in colon cell cancer lines to be transcriptional inactivated by CpG island hypermethylation in the promoter. We therefore examined the DNA methylation state of the Dkk-1 promoter region in Day 26 trophoblast and Day 150 fetal cotyledon tissues comparing AI and SCNT pregnancies. Sequenom MassArray analysis showed very low levels of DNA methylation across the CpG island in the Dkk-1 promoter region of both trophoblast and fetal cotyledon AI tissues. There was no difference in DNA methylation levels between AI and SCNT tissues. Dickkopf-1 is implicated in the success of bovine placental formation as its mRNA expression is down-regulated in SCNT cotyledons. This reduction in gene expression however does not appear to be due to differences in DNA methylation across the CpG island in the promoter region of SCNT tissues when compared with AI controls. |
| Databáze: |
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