Division of Animal Sciences Research Center, University of Missouri, E125 Animal Science Research Center, Columbia, Missouri 65211, USA.
Mol Reprod Dev. 2010 Dec;77(12):1016-30. doi: 10.1002/mrd.21235. Epub 2010 Oct 5.
Nuclear transfer efficiency is low and is thought to be caused by inadequate placental development. The objective of this study was to identify differentially expressed transcripts in pig placentas derived from in vivo fertilization, in vitro fertilization or nuclear transfer at Day 30 of gestation. Three activation methods were compared: electrical fusion/activation, electrical fusion/activation followed by treatment with reversible proteasomal inhibitor, MG132 or electrical fusion followed by activation with Thimerosal/DTT. Extraembryonic membranes were collected 30 days after artificial insemination (IVV) or embryo transfer (IVF and NT). Extraembryonic membrane cDNAs labeled with Cy5 and a reference cDNA labeled with Cy3 were hybridized to a pig reproductive tissue-specific 19,968 spot cDNA microarray. Images acquired and assessed by using Genepix Pro 4.0 were analyzed by Genespring 7.3.1. ANOVA (P < 0.05) identified 227 differentially expressed transcripts between the five treatments and 0 between the three activation methods. The nuclear transfer groups were pooled and compared to in vivo samples, identifying 34 up- and 19 down-regulated transcripts (>2-fold change, P < 0.05). Ten transcripts were validated by real-time PCR. UPTI, PAG2, and GLUD1 protein was quantified by Western blot and densitometry verified that UPTI and PAG2 proteins had an expression pattern that mirrored mRNA abundance (P < 0.05). Localization patterns were also determined for UPTI, PAG2, GLUD2 and 14-3-3 gamma in Day 35 extraembryonic membranes. Observed differences in gene and protein expression in nuclear transfer extraembryonic membranes indicate that an impaired fetal-maternal interface, and not the activation method, may be causing defects observed in cloned pigs.
核转移效率低，据认为是由于胎盘发育不足所致。本研究的目的是鉴定在妊娠第 30 天来自体内受精、体外受精或核转移的猪胎盘中转录本的差异表达。比较了三种激活方法：电融合/激活、电融合/激活后用可逆蛋白酶体抑制剂 MG132 处理或电融合后用硫柳汞/DTT 激活。在人工授精（IVV）或胚胎移植（IVF 和 NT）后 30 天收集胚胎外膜。用 Cy5 标记的胚胎外膜 cDNA 和用 Cy3 标记的参考 cDNA 杂交到猪生殖组织特异性的 19968 个斑点 cDNA 微阵列上。使用 Genepix Pro 4.0 获取和评估图像，并使用 Genespring 7.3.1 进行分析。ANOVA（P<0.05）鉴定了五种处理方法之间的 227 个差异表达转录本，而三种激活方法之间没有差异。将核转移组与体内样本进行合并比较，鉴定出 34 个上调和 19 个下调的转录本（>2 倍变化，P<0.05）。通过实时 PCR 验证了 10 个转录本。通过 Western blot 定量 UP TI、PAG2 和 GLUD1 蛋白，密度计验证 UP TI 和 PAG2 蛋白的表达模式与 mRNA 丰度一致（P<0.05）。还确定了 Day 35 胚胎外膜中 UP TI、PAG2、GLUD2 和 14-3-3 γ的定位模式。在核转移胚胎外膜中观察到的基因和蛋白质表达差异表明，受损的胎儿-母体界面，而不是激活方法，可能是导致克隆猪中观察到的缺陷的原因。
