Improved in vitro development of cloned bovine embryos using S-adenosylhomocysteine, a non-toxic epigenetic modifying reagent.

In this study, fibroblast cells were stably transfected with mouse POU5F1 promoter-driven enhanced green fluorescent protein (EGFP) to investigate the effect of S-adenosylhomocysteine (SAH), the reversible non-toxic inhibitor of DNA-methyltransferases (DNMTs), at different intervals post-fusion on in vitro development of cloned bovine embryos. Treatment with SAH for 12 hr resulted in 54.6 ± 7.7% blastocyst production, which was significantly greater than in vitro fertilized embryos (IVF: 37.2 ± 2.7%), cloned embryos treated with SAH for 72 hr (31.0 ± 7.6%), and control cloned embryos (34.6 ± 3.6%). The fluorescence intensities of the EGFP-POU5F1 reporter gene at all intervals of SAH treatment, except of 72 hr, were significantly higher than control somatic cell nuclear transfers (SCNT) embryos. The intensity of DNA-methylation in cloned embryos treated with SAH for 48 hr was similar to that of IVF embryos, and was significantly lower than the other SCNT groups. The levels of H3K9 acetylation in all SCNT groups were significantly lower than IVF embryos. Real-time PCR analysis of gene expression revealed significantly higher expression of POU5F1 in cloned versus IVF blastocysts. Neither embryo production method (SCNT vs. IVF) nor the SAH treatment interval affected expression of the BCL2 gene. Cloned embryos at all intervals of SAH treatment, except for 24 hr, had significantly increased VEGF transcript compared to IVF and control SCNT embryos. It was suggested that the time interval of DNMT inhibition may have important consequences on different in vitro features of bovine SCNT, and the improving effects of DNMT inhibition on developmental competency of cloned embryos are restricted to a specific period of time preceding de novo methylation.

Jafari S ，Hosseini MS ，Hajian M ，Forouzanfar M ，Jafarpour F ，Abedi P ，Ostadhosseini S ，Abbasi H ，Gourabi H ，Shahverdi AH ，Dizaj AV ，Anjomshoaa M ，Haron W ，Noorshariza N ，Yakub H ，Nasr-Esfahani MH ... -  《-》

Effect of epigenetic modification with trichostatin A and S-adenosylhomocysteine on developmental competence and POU5F1-EGFP expression of interspecies cloned embryos in dog.

Mousai M ，Hosseini SM ，Hajian M ，Jafarpour F ，Asgari V ，Forouzanfar M ，Nasr-Esfahani MH ... -  《-》

Quantitative monitoring of pluripotency gene activation after somatic cloning in cattle.

Wuensch A ，Habermann FA ，Kurosaka S ，Klose R ，Zakhartchenko V ，Reichenbach HD ，Sinowatz F ，McLaughlin KJ ，Wolf E ... -  《BIOLOGY OF REPRODUCTION》

Combination of S-adenosylhomocysteine and scriptaid, a non-toxic epigenetic modifying reagent, modulates the reprogramming of bovine somatic-cell nuclear transfer embryos.

The goal of this study was to improve the development of bovine somatic-cell nuclear transfer (SCNT) embryos by optimizing the combination of DNA methyltransferases inhibitor S-adenosylhomocysteine (SAH) and histone deacetylase inhibitor Scriptaid (SPD). A. 4 × 4-factor design of different drug combinations (0, 0.75, 1.0, and 1.5 mM SAH and 0, 5, 250, and 500 nM SPD) was used to identify an optimal combination of 0.75 mM SAH and 250 nM SPD that improved the developmental competence of bovine SCNT embryos. Further experiments using this combination revealed that methylation levels of CpG islands near exon 1 of the pluripotent gene SOX2; the epigenetic-related gene HDAC3 and DNMT3a; imprinted genes XIST and PEG3; as well as apoptosis-related genes BCL2 and BAX were returned to levels similar to those of in vitro fertilized (IVF) embryo after treatment, which also normalized transcript levels for these genes. This combination also returned global DNA methylation to a normal level, correcting H4K12ac levels while enhancing H3K9ac levels. Thus, the combined application of 0.75 mM SAH and 250 nM SPD can significantly improve the reprogramming of bovine SCNT embryos by stabilizing how embryos utilize their genomes.

Zhang H ，Wang Y ，Sang Y ，Zhang Y ，Hua S ... -  《-》

Cellular composition and viability of cloned bovine embryos using exogene-transfected somatic cells.

Lee SL ，Kumar BM ，Kim JG ，Ock SA ，Jeon BG ，Balasubramanian S ，Choe SY ，Rho GJ ... -  《REPRODUCTION IN DOMESTIC ANIMALS》