Department of Dairy Science, University of Wisconsin-Madison, Madison, Wisconsin, USA.
PLoS One. 2013 Jul 22;8(7):e69490. doi: 10.1371/journal.pone.0069490. Print 2013.
Imprinted genes have been implicated in early embryonic, placental, and neonatal development and alterations in expression levels of these genes can lead to growth abnormalities and embryonic lethality. However, little is known about the functions of bovine imprinted genes during the pre-implantation period. Therefore, the objective of this study was to assess the influence of altered expression of imprinted genes on developmental progress of embryos using small interfering RNA (siRNA). Expression levels of 18 imprinted genes (MAGEL2, UBE3A, IGF2R, NAP1L5, TSSC4, PEG3, NDN, CDKN1C, PHLDA2, MKRN3, USP29, NNAT, PEG10, RTL1, IGF2, H19, MIM1, and XIST) were compared between embryos reaching the blastocyst stage and growth-arrested embryos (degenerates) using quantitative real-time PCR (qRT-PCR). Ten genes were found to be differentially expressed between blastocysts and degenerates. The CDKN1C gene showed the highest upregulation in blastocysts whereas PHLDA2 was highly expressed in degenerates. To assess whether the observed differential gene expression was causative or resultant of embryo degeneration, these genes were selected for functional analysis using siRNA. Injection of siRNA specific to PHLDA2 into one-cell zygotes resulted in a substantial increase in blastocyst development, whereas injection of CDKN1C-specific siRNA resulted in a 45% reduction (P = 0.0006) in blastocyst development. RNA-Seq analysis of CDKN1C-siRNA-injected vs. non-injected embryos revealed 51 differentially expressed genes with functions in apoptosis, lipid metabolism, differentiation, and cell cycle regulation. Gene ontology analysis revealed nine pathways related to cell signaling, metabolism, and nucleic acid processing. Overall, our results show that proper expression levels of the imprinted genes CDKN1C and PHLDA2 are critical for embryo development, which suggests that these genes can be used as markers for normal blastocyst formation.
印记基因参与早期胚胎、胎盘和新生儿的发育，这些基因表达水平的改变可导致生长异常和胚胎致死。然而，关于牛印记基因在胚胎植入前阶段的功能知之甚少。因此，本研究旨在使用小干扰 RNA（siRNA）评估印记基因表达改变对胚胎发育进程的影响。使用定量实时 PCR（qRT-PCR）比较达到囊胚阶段的胚胎和生长停滞的胚胎（退化）之间 18 个印记基因（MAGEL2、UBE3A、IGF2R、NAP1L5、TSSC4、PEG3、NDN、CDKN1C、PHLDA2、MKRN3、USP29、NNAT、PEG10、RTL1、IGF2、H19、MIM1 和 XIST）的表达水平。结果发现，囊胚和退化胚胎之间有 10 个基因差异表达。CDKN1C 基因在囊胚中表达上调最高，而 PHLDA2 在退化胚胎中高表达。为了评估观察到的差异基因表达是胚胎退化的原因还是结果，使用 siRNA 对这些基因进行功能分析。将特异性针对 PHLDA2 的 siRNA 注射到单细胞受精卵中，导致囊胚发育显著增加，而注射 CDKN1C 特异性 siRNA 则导致囊胚发育减少 45%（P = 0.0006）。对 CDKN1C-siRNA 注射与未注射胚胎的 RNA-Seq 分析显示，有 51 个差异表达基因具有凋亡、脂质代谢、分化和细胞周期调节功能。基因本体分析显示，有 9 个与细胞信号转导、代谢和核酸加工相关的途径。总之，我们的研究结果表明，印记基因 CDKN1C 和 PHLDA2 的适当表达水平对胚胎发育至关重要，这表明这些基因可作为正常囊胚形成的标记物。
