Knockdown of CDKN1C (p57(kip2)) and PHLDA2 results in developmental changes in 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 embryonic lethality. However, little is known about the functions of bovine imprinted genes during the pre-implantatio...

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Main Authors: Ashley M Driver, Wen Huang, Jenna Kropp, Francisco Peñagaricano, Hasan Khatib
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2013-01-01
Series:PLoS ONE
Online Access:https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23894493/pdf/?tool=EBI
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spelling doaj-d4097e8f4f5940a0a7e79b0a963484432021-03-03T23:06:48ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-0187e6949010.1371/journal.pone.0069490Knockdown of CDKN1C (p57(kip2)) and PHLDA2 results in developmental changes in bovine pre-implantation embryos.Ashley M DriverWen HuangJenna KroppFrancisco PeñagaricanoHasan KhatibImprinted 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.https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23894493/pdf/?tool=EBI
collection DOAJ
language English
format Article
sources DOAJ
author Ashley M Driver
Wen Huang
Jenna Kropp
Francisco Peñagaricano
Hasan Khatib
spellingShingle Ashley M Driver
Wen Huang
Jenna Kropp
Francisco Peñagaricano
Hasan Khatib
Knockdown of CDKN1C (p57(kip2)) and PHLDA2 results in developmental changes in bovine pre-implantation embryos.
PLoS ONE
author_facet Ashley M Driver
Wen Huang
Jenna Kropp
Francisco Peñagaricano
Hasan Khatib
author_sort Ashley M Driver
title Knockdown of CDKN1C (p57(kip2)) and PHLDA2 results in developmental changes in bovine pre-implantation embryos.
title_short Knockdown of CDKN1C (p57(kip2)) and PHLDA2 results in developmental changes in bovine pre-implantation embryos.
title_full Knockdown of CDKN1C (p57(kip2)) and PHLDA2 results in developmental changes in bovine pre-implantation embryos.
title_fullStr Knockdown of CDKN1C (p57(kip2)) and PHLDA2 results in developmental changes in bovine pre-implantation embryos.
title_full_unstemmed Knockdown of CDKN1C (p57(kip2)) and PHLDA2 results in developmental changes in bovine pre-implantation embryos.
title_sort knockdown of cdkn1c (p57(kip2)) and phlda2 results in developmental changes in bovine pre-implantation embryos.
publisher Public Library of Science (PLoS)
series PLoS ONE
issn 1932-6203
publishDate 2013-01-01
description 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.
url https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23894493/pdf/?tool=EBI
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