Amino acid supplementation affects imprinted gene transcription patterns in parthenogenetic porcine blastocysts.

To determine whether exogenous amino acids affect gene transcription patterns in parthenogenetic porcine embryos, we investigated the effects of amino acid mixtures in culture medium. Parthenogenetic embryos were cultured in PZM3 medium under four experimental conditions: 1) control (no amino acids except L-glutamine and taurine); 2) nonessential amino acids (NEAA); 3) essential amino acids (EAA); and 4) NEAA and EAA. The rate of development of embryos to the four-cell stage was not affected by treatment. However, fewer (P<0.05) embryos cultured with EAA (12.8%) reached the blastocyst stage as compared with the control group (25.6%) and NEAA group (30.3%). Based on these findings, we identified genes with altered expression in parthenogenetic embryos exposed to medium with or without EAAs. The results indicated that EAA influenced gene expression patterns, particularly those of imprinted genes (e.g., H19, IGF2R, PEG1, XIST). However, NEAAs did not affect impaired imprinted gene expressions induced by EAA. The results also showed that mechanistic target of rapamycin (MTOR) mRNA expression was significantly increased by EAA alone as compared with control cultures, and that the combined treatment with NEAA and EAA did not differ significantly from those of control cultures. Our results revealed that gene transcription levels in porcine embryos changed differentially depending on the presence of EAA or NEAA. However, the changes in the H19 mRNA observed in the parthenogenetic blastocysts expression level was not related to the DNA methylation status in the IGF2/H19 domain. The addition of exogenous amino acid mixtures affected not only early embryonic development, but also gene transcription levels, particularly those of imprinted genes. However, this study did not reveal how amino acids affect expression of imprinted genes under the culture conditions used. Further studies are thus required to fully evaluate how amino acids affect transcriptional regulation in porcine embryos

X-linked gene transcription patterns in female and male in vivo, in vitro and cloned porcine individual blastocysts.

To determine the presence of sexual dimorphic transcription and how in vitro culture environments influence X-linked gene transcription patterns in preimplantation embryos, we analyzed mRNA expression levels in in vivo-derived, in vitro-fertilized (IVF), and cloned porcine blastocysts. Our results clearly show that sex-biased expression occurred between female and male in vivo blastocysts in X-linked genes. The expression levels of XIST, G6PD, HPRT1, PGK1, and BEX1 were significantly higher in female than in male blastocysts, but ZXDA displayed higher levels in male than in female blastocysts. Although we found aberrant expression patterns for several genes in IVF and cloned blastocysts, similar sex-biased expression patterns (on average) were observed between the sexes. The transcript levels of BEX1 and XIST were upregulated and PGK1 was downregulated in both IVF and cloned blastocysts compared with in vivo counterparts. Moreover, a remarkable degree of expression heterogeneity was observed among individual cloned embryos (the level of heterogeneity was similar in both sexes) but only a small proportion of female IVF embryos exhibited variability, indicating that this phenomenon may be primarily caused by faulty reprogramming by the somatic cell nuclear transfer (SCNT) process rather than in vitro conditions. Aberrant expression patterns in cloned embryos of both sexes were not ameliorated by treatment with Scriptaid as a potent HDACi, although the blastocyst rate increased remarkably after this treatment. Taken together, these results indicate that female and male porcine blastocysts produced in vivo and in vitro transcriptional sexual dimorphisms in the selected X-linked genes and compensation of X-linked gene dosage may not occur at the blastocyst stage. Moreover, altered X-linked gene expression frequently occurred in porcine IVF and cloned embryos, indicating that X-linked gene regulation is susceptible to in vitro culture and the SCNT process, which may eventually lead to problems with embryonic or placental defects

Production of multiple transgenic Yucatan miniature pigs expressing human complement regulatory factors, human CD55, CD59, and H-transferase genes.

The present study was conducted to generate transgenic pigs coexpressing human CD55, CD59, and H-transferase (HT) using an IRES-mediated polycistronic vector. The study focused on hyperacute rejection (HAR) when considering clinical xenotransplantation as an alternative source for human organ transplants. In total, 35 transgenic cloned piglets were produced by somatic cell nuclear transfer (SCNT) and were confirmed for genomic integration of the transgenes from umbilical cord samples by PCR analysis. Eighteen swine umbilical vein endothelial cells (SUVEC) were isolated from umbilical cord veins freshly obtained from the piglets. We observed a higher expression of transgenes in the transgenic SUVEC (Tg SUVEC) compared with the human umbilical vein endothelial cells (HUVEC). Among these genes, HT and hCD59 were expressed at a higher level in the tested Tg organs compared with non-Tg control organs, but there was no difference in hCD55 expression between them. The transgenes in various organs of the Tg clones revealed organ-specific and spatial expression patterns. Using from 0 to 50% human serum solutions, we performed human complement-mediated cytolysis assays. The results showed that, overall, the Tg SUVEC tested had greater survival rates than did the non-Tg SUVEC, and the Tg SUVEC with higher HT expression levels tended to have more down-regulated α-Gal epitope expression, resulting in greater protection against cytotoxicity. By contrast, several Tg SUVEC with low CD55 expression exhibited a decreased resistance response to cytolysis. These results indicated that the levels of HT expression were inversely correlated with the levels of α-Gal epitope expression and that the combined expression of hCD55, hCD59, and HT proteins in SUVECs markedly enhances a protective response to human serum-mediated cytolysis. Taken together, these results suggest that combining a polycistronic vector system with SCNT methods provides a fast and efficient alternative for the generation of transgenic large animals with multiple genetic modifications

Effects of Trichostatin A on Development of Porcine Embryos Derived from Somatic Cell Nuclear Transfer

Many different approaches have been developed to improve the efficiency of animal cloning by somatic cell nuclear transfer (SCNT), one of which is to modify histone acetylation levels using histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA). In the present study, we examined the effect of TSA on in vitro development of porcine embryos derived from SCNT. We found that TSA treatment (50 nM) for 24 h following oocyte activation improved blastocyst formation rates (to 22.0%) compared with 8.9% in the non-treatment group and total cell number of the blastocysts for determining embryo quality also increased significantly (88.9→114.4). Changes in histone acetylation levels as a result of TSA treatment were examined using indirect immunofluorescence and confocal microscopy scanning. Results showed that the histone acetylation level in TSA-treated embryos was higher than that in controls at both acetylated histone H3 lysine 9 (AcH3K9) and acetylated histone H4 lysine 12 (AcH4K12). Next, we compared the expression patterns of seven genes (OCT4, ID1; the pluripotent genes, H19, NNAT, PEG1; the imprinting genes, cytokeratin 8 and 18; the trophoblast marker genes). The SCNT blastocysts both with and without TSA treatment showed lower levels of OCT4, ID1, cytokeratin 8 and 18 than those of the in vivo blastocysts. In the case of the imprinting genes H19 and NNAT, except PEG1, the SCNT blastocysts both with and without TSA treatment showed higher levels than those of the in vivo blastocysts. Although the gene expression patterns between cloned blastocysts and their in vivo counterparts were different regardless of TSA treatment, it appears that several genes in NT blastocysts after TSA treatment showed a slight tendency toward expression patterns of in vivo blastocysts. Our results suggest that TSA treatment may improve preimplantation porcine embryo development following SCNT

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

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

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