Knockdown of CDKN1C (p57kip2) and PHLDA2 Results in Abnormal Development of Bovine Pre-Implantation Embryos

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 embryo lethality. However, limited information is known about the functions of the 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 embryo quality using small interfering RNA (siRNA). Expression levels of 17 imprinted genes 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 significant expression in blastocysts whereas PHLDA2 was highly expressed in degenerates. To assess whether the observed differential expression was causative or resultant of embryo degeneration, these genes were selected for functional analysis using siRNA. Injection of siRNA specific to PHLDA2 into a one-cell zygotes resulted in a significant increase in blastocyst development, whereas injection of CDKN1C-specific siRNA resulted in 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 markers for normal blastocyst formation.