Transcriptome profiling of rabbit parthenogenetic blastocysts developed under in vivo conditions

Parthenogenetic embryos are one attractive alternative as a source of embryonic stem cells, although many aspects related to the biology of parthenogenetic embryos and parthenogenetically derived cell lines still need to be elucidated. The present work was conducted to investigate the gene expression profile of rabbit parthenote embryos cultured under in vivo conditions using microarray analysis. Transcriptomic profiles indicate 2541 differentially expressed genes between parthenotes and normal in vivo fertilised blastocysts, of which 76 genes were upregulated and 16 genes downregulated in in vivo cultured parthenote blastocyst, using 3 fold-changes as a cut-off. While differentially upregulated expressed genes are related to transport and protein metabolic process, downregulated expressed genes are related to DNA and RNA binding. Using microarray data, 6 imprinted genes were identified as conserved among rabbits, humans and mice: GRB10, ATP10A, ZNF215, NDN, IMPACT and SFMBT2. We also found that 26 putative genes have at least one member of that gene family imprinted in other species. These data strengthen the view that a large fraction of genes is differentially expressed between parthenogenetic and normal embryos cultured under the same conditions and offer a new approach to the identification of imprinted genes in rabbit. © 2012 Naturil-Alfonso et al.This work was supported by Generalitat Valenciana research programme (Prometeo 2009/125). Carmen Naturil was supported by Generalitat Valenciana research programme (Prometeo 2009/125). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Naturil Alfonso, C.; Saenz De Juano Ribes, MDLD.; Peñaranda, D.; Vicente Antón, JS.; Marco Jiménez, F. (2012). Transcriptome profiling of rabbit parthenogenetic blastocysts developed under in vivo conditions. PLoS ONE. 7(12):1-11. https://doi.org/10.1371/journal.pone.0051271S111712Harness, J. 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Classification of differentially expressed transcript probes based on fold changes.

<p>Classification of differentially expressed transcript probes based on fold changes.</p

Genes upregulated by at least three-fold in parthenogenetic late blastocysts.

<p>Genes are tabulated in the descending order of the fold-change values. Transcripts without annotation were identified by probe set ID.</p

Genes downregulated by at least three-fold in parthenogenetic late blastocysts.

<p>Genes are tabulated in the descending order of the fold-change values. Transcripts without annotation were identified by probe set ID.</p

Gene Ontology (GO) bar chart of differentially expressed genes between parthenotes and fertilised embryos.

<p>Gene Ontology (GO) bar chart of differentially expressed genes between parthenotes and in vivo fertilised embryos. Genes upregulated and downregulated in parthenotes embryos that are categorised by GO term “Biological process” level 4.</p

Principal Component Analysis (PCA) of microarray data.

<p>Principal Component Analysis (PCA) of microarray data. PCA two-dimensional scatter plot represent the differential gene expression patterns of frozen and control embryos. Axis: X = PC1: PCA Component 1 (56.75% variance); Y = PC2: PCA Component 2 (18.17% variance).</p

Gene Ontology (GO) bar chart of differentially expressed genes between parthenotes and fertilised embryos.

<p>Gene Ontology (GO) bar chart of differentially expressed genes between parthenotes and in vivo fertilised embryos. Genes upregulated and downregulated in parthenotes embryos that are categorised by GO term “Molecular function” level 4.</p

Real-time quantitative PCR assay for six randomly selected genes.

<p><i>SMARCA2</i>: SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 2; <i>EMP1</i>: Epithelial membrane protein 1; <i>CALC</i>: calcitonin gene-related peptide variant 1; <i>SCGB1A1</i>: secretoglobin family 1A member 1). Relative expression values are shown in arbitrary units (a.u), expressed by the mean value ± standard error means. Letters with different superscripts are significantly different (P<0.05). RT-qPCR fold changes were obtained by calculation of log2 transformed ratio of relative expression for each gene. Microarray fold changes were obtained by log2 transformed probe intensities for each gene.</p

Putative imprinted genes differentially expressed in parthenogenetic late blastocysts identified as family members at Catalogue of Imprinted Genes (http://igc.otago.ac.nz/home.html).

<p>Putative imprinted genes differentially expressed in parthenogenetic late blastocysts identified as family members at Catalogue of Imprinted Genes (<a href="http://igc.otago.ac.nz/home.html" target="_blank">http://igc.otago.ac.nz/home.html</a>).</p

Gene Ontology (GO) bar chart of differentially expressed genes between parthenotes and fertilised embryos.

<p>Gene Ontology (GO) bar chart of differentially expressed genes between parthenotes and in vivo fertilised embryos. Genes upregulated and downregulated in parthenotes embryos that are categorised by GO term “Cellular Component” level 7.</p