Kohan-Ghadr H R, Smith L C, Arnold D R, Murphy B D, Lefebvre R C
Department of Clinical Sciences, University of Montreal, 3200 Sicotte, Saint-Hyacinthe, Québec J2S 2M2, Canada.
Reprod Fertil Dev. 2012;24(4):588-98. doi: 10.1071/RD11162.
Abnormal placental development is common in the bovine somatic cell nuclear transfer (SCNT)-derived fetus. In the present study, we characterised the expression of E-cadherin and β-catenin, structural proteins of adherens junctions, in SCNT gestations as a model for impaired placentation. Cotyledonary tissues were separated from pregnant uteri of SCNT (n = 6) and control pregnancies (n = 8) obtained by artificial insemination. Samples were analysed by western blot, quantitative RT-PCR (qRT-PCR) and immunohistochemistry. Bovine trophectoderm cell lines derived from SCNT and control embryos were analysed to compare with the in utero condition. Although no differences in E-cadherin or β-catenin mRNA abundance were observed in fetal tissues between the two groups, proteins encoded by these genes were markedly under-expressed in SCNT trophoblast cells. Immunohistochemistry revealed a different pattern of E-cadherin and total β-catenin localisation in SCNT placentas compared with controls. No difference was observed in subcellular localisation of dephosphorylated active-β-catenin protein in SCNT tissues compared with controls. However, qRT-PCR confirmed that the wingless (WNT)/β-catenin signalling pathway target genes CCND1, CLDN1 and MSX1 were downregulated in SCNT placentas. No differences were detected between two groups of bovine trophectoderm cell lines. Our results suggest that impaired expression of E-cadherin and β-catenin proteins, along with defective β-catenin signalling during embryo attachment, specifically during placentation, is a molecular mechanism explaining insufficient placentation in the bovine SCNT-derived fetus.
异常胎盘发育在牛体细胞核移植（SCNT）衍生的胎儿中很常见。在本研究中，我们将SCNT妊娠中粘着连接的结构蛋白E-钙粘蛋白和β-连环蛋白的表达特征作为胎盘形成受损的模型进行研究。从通过人工授精获得的SCNT妊娠（n = 6）和对照妊娠（n = 8）的怀孕子宫中分离出子叶组织。通过蛋白质免疫印迹、定量逆转录聚合酶链反应（qRT-PCR）和免疫组织化学分析样本。分析源自SCNT和对照胚胎的牛滋养外胚层细胞系以与子宫内情况进行比较。尽管两组胎儿组织中未观察到E-钙粘蛋白或β-连环蛋白mRNA丰度的差异，但这些基因编码的蛋白质在SCNT滋养层细胞中明显低表达。免疫组织化学显示，与对照组相比，SCNT胎盘组织中E-钙粘蛋白和总β-连环蛋白的定位模式不同。与对照组相比，SCNT组织中去磷酸化活性β-连环蛋白的亚细胞定位未观察到差异。然而，qRT-PCR证实，无翅（WNT）/β-连环蛋白信号通路靶基因CCND1、CLDN1和MSX1在SCNT胎盘中下调。两组牛滋养外胚层细胞系之间未检测到差异。我们的结果表明，E-钙粘蛋白和β-连环蛋白的表达受损，以及胚胎着床期间，特别是胎盘形成期间β-连环蛋白信号缺陷，是解释牛SCNT衍生胎儿胎盘形成不足的分子机制。
