Our objective is to investigate the effect of valproic acid (VPA), a histone deacetylase inhibitor, on early embryonic development. We studied the effect of VPA on the in vitro maturation of bovine oocytes, and on the development of bovine embryos derived from in vitro fertilization (IVF) or parthenogenesis. Germinal vesicle stage bovine oocytes were cultured with different concentrations of VPA for 24 h; low dose VPA treatment (0.03 and 0.3 mM) had no effect on oocyte maturation, but 3 and 6 mM VPA significantly decreased maturation rate; when used for IVF or parthenogenesis, VPA-treated oocytes generated significantly lowered blastocyst rate. Oocytes matured in vitro were fertilized or underwent parthenogenetic activation; 6 h later, they were exposed to VPA for 48 h, and then the cleavage rate, blastocyst rate and mRNA expression levels of transcription factors (Oct4, Nanog, and Cdx2) were assessed. For embryos cultured in 0.3 mM VPA, there was no remarkable change in cleavage rate or blastocyst rate, but the expression of Oct4 and Nanog in blastocysts was significantly increased. For embryos treated with 3.0 mM VPA, the cleavage rate and blastocyst rate were significantly decreased. In conclusion, low dose VPA has no effect on oocyte maturation but affects subsequent embryonic development. Low dose VPA administration to IVF embryos had no effect on embryonic development, but the expression of several important transcription factors was increased. Treatment of IVF embryos with low dose VPA may improve their development potential.

Transmembrane protein 98 (TMEM98) is a recently discovered gene, the inhibition of which has preliminarily been demonstrated to inhibit progression of several solid cancers in vitro. However, its involvement in tumorigenesis of gastric cancer (GC) has not been reported. Here, we aimed to explore the expression of TMEM98 in GC tissues and cell lines and to determine the role of TMEM98 in GC cell proliferation and invasion. TMEM98 was significantly upregulated in GC tissues, which was associated with low survival rate of GC patients. Interestingly, GC cell proliferation and invasion were promoted by TMEM98 messenger RNA (mRNA) upregulation and inhibited by TMEM98 mRNA downregulation, but not affected by TMEM98 protein. Using RNA‐binding protein immunoprecipitation assay and RNA pull‐down assay, we demonstrated that TMEM98 mRNA could directly bind with and upregulate nuclear factor 90 (NF90). Similarly, NF90 protein could not only enhance the stability of TMEM98 mRNA but antagonize the suppressive effect of TMEM98‐small interfering RNA on proliferation and invasion in MKN‐45 cells. Moreover, RNA pull‐down assay, with wild‐type (WT) and binding‐site‐mutated biotinylated TMEM98 mRNA transcripts, demonstrated that WT TMEM98 mRNA bound with NF90 protein through an 8‐nt motif at the last exon, but the motif mutation abolished the capacity of TMEM98 mRNA binding to NF90 protein. Furthermore, overexpression of the WT last exon of TMEM98 increased NF90 expression and cell proliferation/invasion expectedly, but overexpression of the mutated last exon had no obvious effect. In conclusion, TMEM98 mRNA enhanced the proliferation and invasion of GC cells by interacting with the NF90 protein.

DNA methylation in mammals is an epigenetic marker and necessary for normal embryogenesis. The global genomic demethylation of 5-methylcytosine occurs during the first cell cycle following fertilization. Activation-induced cytidine deaminase (AID), which is well-known for the function in antibody diversification, has been implicated to play a role in active demethylation, but its role in cell reprogramming and its crosstalk with other DNA demethylation mechanism need to be clarified. In this study, the dynamic epigenetic regulation of cell pluripotency and embryo development by AID in bovine preimplantation embryos was investigated. The analysis of an AID overexpressing transgenic cell line showed that AID overexpression did not change the global genomic methylation but did change the methylation status of the promoters of the OCT4, NANOG and SOX2 genes, thereby causing changes in their expression. The siRNA-mediated AID knockdown in early embryonic development indicated that AID interference did not affect oocyte maturation or the following embryo development after in vitro fertilization but influenced the DNA methylation status of OCT4 and NANOG. To clarify the role of AID in preimplantation embryos, SCNT embryos were obtained using AID-overexpressing cells as nuclear donors. Compared to the control group, the cleavage and blastocyst rates were both significantly improved in the AID-overexpression group. The expression of OCT4 and NANOG was increased in the SCNT embryos, whereas the methylation levels of their promoters were reduced. In conclusion, this study demonstrated that AID selectively catalyzes DNA demethylation of pluripotency genes to play a role in regulation their expression, improves bovine SCNT embryo development by increased expression levels.

SCNT technology has been successfully used to clone a variety of mammals, but the cloning efficiency is very low. This low efficiency is likely due to the incomplete reprogramming of SCNT embryos. Histone modification and DNA methylation may participate in these events. Thus, it would be interesting to attempt to improve the efficiency of SCNT by using a HDACi VPA. In order to guarantee the effect of VPA and reduce its cytotoxicity, a comprehensive analysis of the cell proliferation and histone modification was performed. The results showed that 0.5 and 1 mM VPA treatment for 24 h were the optimal condition. According to the results, H3K4me3 was increased in 0.5 and 1 mM VPA groups, whereas H3K9me2 was significantly decreased. These are the signals of gene-activation. In addition, VPA treatment led to the overexpression of Oct4 and Nanog. These indicated that VPA-treated cells had similar patterns of histone to zygotic embryos, and may be more favorable for reprograming. A total of 833 cloned embryos were produced from the experimental replicates of VPA-treated donor cells. In 1 mM treatment group, the blastocyst rates were significantly increased compared with control. At the same time, our findings demonstrated the interrelation between DNA methylation and histone modifications.