Real-time PCR analysis of mRNA expression levels in parthenogenetic blastocysts cultured with/without EAAs.

<p>The relative mRNA expression of the selected 15 genes; <i>HKII</i>, <i>ACACA</i>, <i>PCmt</i>, <i>MCT1</i>, <i>SLC3A1</i>, <i>GLUT2</i>, <i>GRB10</i>, <i>H19</i>, <i>IGF2R</i>, <i>NNAT</i>, <i>PEG1</i>, <i>XIST</i>, <i>BEX1</i>, <i>G6PD</i>, and <i>PGK1</i>, in EAA blastocysts (<i>n</i> = 30) after normalization relative to the average of <i>RN18S</i> and <i>ACTB</i> (internal control) gene, were compared to those of the blastocysts cultured in the absence of amino acid mixtures (control) which was set to 1. Bars with asterisks indicate significantly different from the value in the control (*, <i>P</i><0.05). Data is mean ± S.E.M.</p

Methylation status of <i>IGF2/H19</i> DMR3 in porcine parthenogenetic blastocysts.

<p>The methylation patterns of DMR3 in porcine: A, adult liver tissue; B, control parthenogenetic blastocysts (<i>n</i> = 50); and C, blastocysts cultured with EAA (<i>n</i> = 50). Individual circles indicate a CpG dinucleotide. Open and solid circles represent unmethylated and methylated CpGs, respectively.</p

Effects of amino acids on the development of parthenogenetic embryos.^†

†<p>The number of replicates was 10. The cleavage and blastocyst rates were recorded 48 h and 144 h post-activation, respectively. All percentage data expressed show mean values ± SEM. The asterisk indicates a significant difference determined by Analysis of Variance (<i>P</i><0.05).</p><p>Effects of amino acids on the development of parthenogenetic embryos.^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106549#nt102" target="_blank">†</a>}</p

mRNA expression pattern of <i>H19</i> and <i>IGF2R</i> in individual parthenogenetic blastocysts.

<p>The quantitative data represents the values from transcripts of <i>H19</i> and <i>IGF2R</i> genes in individual mRNA samples (<i>n</i> = 20). Bars with asterisks indicate significantly different from the value in the control (*, <i>P</i><0.05). Data is mean ± S.E.M.</p

Differential mRNA expression patterns for four imprinted genes in parthenogenetic blastocysts cultured with NEAA or EAA or both.

<p>Bars (A) and aligned dot plots (B) represent mRNA transcript levels of the imprinted <i>H19</i>, <i>IGF2R</i>, <i>NNAT</i> and <i>PEG1</i> genes in individual blastocysts after normalization relative to the average of <i>RN18S</i> and <i>ACTB</i> (internal control) genes, were compared to those of control blastocysts cultured in the absence of amino acid mixture, which was set to 1. The aligned dot plots with asterisks indicate significantly different from 1 (*, <i>P</i><0.05; **, <i>P</i><0.001; ***, <i>P</i><0.0001).</p

Total cell numbers of parthenogenetic blastocysts cultured with NEAA or EAA or both.

<p>Scatter dot plots represent average numbers of total cells in control (41.6; <i>n</i> = 48), NEAA (42.2; <i>n</i> = 48), EAA^D3 (32.7 <i>n</i> = 37), EAA^D6 (30.8; <i>n</i> = 36), EAA^half (31.3; <i>n</i> = 41), and NEAA + EAA (40.5; <i>n</i> = 45) blastocysts at Day 6. Cells were counted under UV using a fluorescence microscope with a DAPI fluorescence emission filter in three independent experiments. <i>P</i>-values are derived when treatments are compared to control (*, <i>P</i><0.05, **, <i>P</i><0.001).</p

<i>MTOR</i> mRNA expression in parthenogenetic blastocysts cultured with NEAA or EAA or both.

<p>The values from transcripts of <i>MTOR</i> gene in individual samples of each group (<i>n</i> = 12) are expressed as mean ± S.E.M. The means of each AA treatment group were compared to those of control blastocysts. Bars with asterisks indicate significant difference between in control and amino acid treatment groups (**, <i>P</i><0.001).</p

<i>H19</i> and <i>IGF2R</i> mRNA levels between the amino acid treatments after 48 h of culture and for whole embryo culture.

<p>The embryos (NEAA/EAA^D3) were cultured with NEAAs for the first 48 h. Then, these embryos were transferred and subsequently cultured in PZM3 supplemented with NEAA + EAA up to 144 h. The quantitative data represents the values from transcripts of <i>H19</i>: A and <i>IGF2R</i>: B genes in individual mRNA samples (*, <i>P</i><0.05; **, <i>P</i><0.001).</p