Induced DNA hypomethylation by Folic Acid Deprivation in Bovine Fibroblast Donor Cells Improves Reprogramming of Somatic Cell Nuclear Transfer Embryos.

Aberrant patterns of DNA methylation are consistent events in SCNT derived embryos and mechanistically are believed to be related to abnormal development. While some epigenetic drugs have been used in attempts to improve SCNT efficiency but some concerns remained toward the safety of these drugs on the health of future offspring. Folate is an essential cofactor in one-carbon cycle for conversion of homocysteine to methionine, thereby ensuring supply of SAM, the universal methyl donor for many biological methylation reactions including DNA methylation. Therefore, in vitro DNA hypo-methylation can be induced by folate deprivation and this study aims at deciphering the role of folic acid deprivation in culture medium of BFFs for 6 days on SCNT efficiency. Our data revealed that culture of fibroblast cells in folate- medium containing 0.5% FBS did not alter the cell cycle compared to other groups. Flowcytometric analysis revealed that DNA methylation (5-mC level) in folate deprived cells cultured in 0.5% serum was decreased compared to folate+ group. The result of bisulfite sequencing was in accordance with flowcytometric analysis, which indicated a decrease in DNA methylation of POU5F1 promoter. Gene expression analysis revealed an increase in expression of POU5F1 gene in folate- group. The nuclear area of the cells in folate- group was significantly larger than folate+ group. Induced DNA hypomethylation by folate deprivation in the folate- group significantly improved blastocyst rate compared to the folate+ group. DNA methylation level in POU5F1 promoter and ICR of H19 and IGF2 of SCNT derived embryos in the folate- group was similar to the IVF derived blastocysts. In conclusion, our results proposes a promising "non-chemical" instead of "chemical" approach using inhibitors of epigenetic modifier enzymes for improving mammalian SCNT efficiency for agricultural and biomedical purposes.

Functional characterization of NANOG in goat pre-implantation embryonic development.

Nanog as a novel pluripotent cell-specific gene plays important roles in regulation of signaling pathways for maintenance and induction of pluripotency in inner cell mass (ICM) and embryonic stem cells (ESC) in mouse. The molecular features and transcription regulation of NANOG gene in domestic animals are not well defined. In this study, we performed knockdown of NANOG mRNA in goat embryos and examined its effect on early embryonic development. Presumptive zygotes were injected with a volume of 8-10 pl NANOG or scrambled (SCR) siRNA, and subsequently cleavage and blastocyst formation rate were assessed. Furthermore, gene expression analysis was carried out in 6-8 cell and blastocyst derived embryos from non-injected controls, SCR - and siRNA-injected presumptive zygotes. Cleavage and blastocyst rates in siRNA groups were insignificantly lower than the control and SCR groups. Embryos with reduced expression of NANOG showed decrease in number of trophectoderm (TE) and total cells in blastocysts. Analysis of expression of developmentally important genes (SOX2, OCT4 and NANOG), which work as a network, showed that NANOG knockdown results in significant increase in expression of SOX2 and OCT4 and among the possible target genes (CDX2, REX1 and GATA4) of this network, only GATA4 showed increased expression. Our results suggest that NANOG is likely to be required for proliferation of trophoblastic cells.

Urea changes oocyte competence and gene expression in resultant bovine embryo in vitro.

SummaryNutrition influences the microenvironment in the proximity of oocyte and affects early embryonic development. Elevated blood urea nitrogen, even in healthy dairy cows, is associated with reduced fertility and there is high correlation between blood urea levels and follicular fluid urea levels. Using a docking calculation (in silico), urea showed a favorable binding activity towards the ZP-N domain of ZP3, that of ZP2, and towards the predicted full-length sperm receptor ZP3. Supplementation of oocyte maturation medium with nutrition-related levels of urea (20 or 40 mg/dl as seen in healthy dairy cows fed on low or high dietary protein, respectively) dose-dependently increased: (i) the proportion of oocytes that remained uncleaved; and (ii) oocyte degeneration; and reduced cleavage, blastocyst and hatching rates. High levels of urea induced shrinkage in oocytes, visualised using scanning electron microscopy. Urea downregulated NANOG while dose-dependently upregulating OCT4, DNMT1, and BCL2 expression. Urea at 20 mg/dl induced BAX expression. Using mathematical modelling, the rate of oocyte degeneration was sensitive to urea levels; while cleavage, blastocyst and hatching rates exhibited negative sensitivity. The present data imply a novel role for urea in reducing oocyte competence and changing gene expression in the resultant embryos.

Cardiac differentiation of mouse embryonic stem cells is influenced by a PPAR γ/PGC-1α-FNDC5 pathway during the stage of cardiac precursor cell formation.

Peroxisome proliferator-activated receptor (PPAR) γ co-activator 1α (PGC-1α) up-regulation induces FNDC5 expression in muscle and consequently causes browning of white adipose tissue (WAT). In addition to skeletal muscle, FNDC5 is mainly expressed in heart and brain tissues. Here, we demonstrate that FNDC5 expression increased during the process of cardiac differentiation of mouse embryonic stem cells (mESCs) similar to PGC-1α and PPARα. To testify the correlation between PGC-1α and FNDC5 in cardiac cell differentiation of mESCs, we utilized specific PPARγ agonist and antagonist in two stages of cardiac differentiation, during and post-cardiac precursor cells (CPCs) formation. Our results indicated that a reduction in PGC-1α expression, via treatment with GW9662 during CPCs formation stage, down-regulated FNDC5 transcript levels as well as mitochondrial markers which negatively influenced on the whole process of cardiac differentiation efficiency. On the other hand, increase PGC-1α expression during CPCs formation stage via rosiglitazone treatment increase FNDC5 and mitochondrial markers transcript levels which enhanced cardiac differentiation efficiency. Importantly, such alteration in PGC-1α expression at post-CPCs formation stage did not affect overall cardiac differentiation rate as expression of FNDC5 and mitochondrial markers were not significantly changed. We concluded that PPARγ agonist and antagonist induced up and down-regulation of PGC-1α and subsequently modulated the process of CPCs formation through an alteration in FNDC5 and mitochondrial markers expression.