RNAi-mediated knockdown of Xist improves development of the female buffalo (Bubalus bubalis) nuclear transfer embryos.

Xist plays a critical role in the X-chromosome inactivation (XCI), an important epigenetic reprogramming of somatic cell nuclear transfer (SCNT) embryos. Modulation of Xist expression enhanced the developmental ability of mouse cloned embryos. However, the roles of Xist in buffalo SCNT embryos remain unknown. In this study, we investigated the methylation and expression status of Xist in different genders of buffalo donor cells and various stages (two-cell, eight-cell, morula and blastocyst) of in vitro fertilization (IVF) and SCNT embryos. The methylation of Xist in SCNT-♀ and SCNT-♂ embryos was aberrant hypomethylation compared with the buffalo foetal fibroblast (♀-BFF and ♂-BFF), IVF-♀ and IVF-♂ embryos. At the eight-cell stage, Xist expression was significantly higher in SCNT-♀ embryos compared with those in SCNT-♂, IVF-♀ and IVF-♂ embryos (P < 0.05). Meanwhile, no significant difference was found between IVF-♀ and IVF-♂ embryos (P > 0.05). Accordingly, we suppressed Xist expression by RNAi-Xist in SCNT-♀ embryos. Results showed that injection of Xist-shRNA significantly improved the morula and blastocyst rates (P < 0.05). These results indicated that correcting the abnormal expression of the Xist gene contributed to the development of SCNT-♀ embryos.

Transforming Growth Factor-ß1 Enhances Mesenchymal Characteristics of Buffalo (Bubalus bubalis) Bone Marrow-Derived Mesenchymal Stem Cells.

Bone marrow-derived mesenchymal stem cells (BMSCs) from livestock are valuable resources for animal reproduction and veterinary therapeutics. Previous studies have shown that BMSCs were prone to malignant transformation of mesenchymal-to-epithelial transition in vitro, which can cause many barriers to further application of BMSCs. The transforming growth factor ß (TGF-ß) signaling pathway has been widely studied as the most important signaling pathway involved in regulating mesenchymal features of BMSCs. However, the effects of the TGF-ß signaling pathway on mesenchymal characteristics of buffalo BMSCs (bBMSCs) remain unclear. In the present study, the impacts of the growth factor, TGF-ß1, on cell proliferation, apoptosis, migration, and karyotype of bBMSCs were tested. Besides, the effects of TGF-ß1 on pluripotency, mesenchymal markers, and epithelial-to-mesenchymal transition (EMT)-related gene expression of bBMSCs were also examined. Results showed that the suitable concentration and time of TGF-ß1 treatment (2 ng/mL and 24 hours) promoted cell proliferation and significantly reduced cell apoptosis (p < 0.05) in bBMSCs. The cell migration capacity and normal karyotype rate of bBMSCs were significantly (p < 0.05) improved under TGF-ß1 treatment. The expression levels of pluripotency-related genes (Sox2 and Nanog) and mesenchymal markers (N-cadherin and Fn1) were significantly (p < 0.05) up-regulated under TGF-ß1 treatment. Furthermore, TGF-ß1 activated the EMT process, thereby contributing to significantly enhancing the expression levels of EMT-related genes (Snail and Slug) (p < 0.05), which in turn improved maintenance of the mesenchymal nature in bBMSCs. Finally, bBMSCs underwent self-transformation more easily and efficiently and exhibited more characteristics of mesenchymal stem cells under TGF-ß1 treatment. This study provides theoretical guidance for elucidating the detailed mechanism of the TGF-ß signaling pathway in mesenchymal feature maintenance of bBMSCs and is of significance to establish a stable culture system of bBMSCs.

The Role of 5-aza-2'-Deoxycytidine on Methylation Status of Xist Gene in Different Genders of Buffalo (Bubalus bubalis) Bone Marrow Mesenchymal Stem Cells.

Previous studies have demonstrated that proper concentration of 5-aza-2'-deoxycytidine (5-aza-CdR) treatment was advantageous to decrease DNA methylation level, but the relationships between 5-aza-CdR treatment and methylation status of imprinted genes are seldom detected. The aim of this study was to investigate the effect of low concentration 5-aza-CdR treatment on the methylation status of imprinted gene Xist in different genders of buffalo bone marrow mesenchymal stem cells (BMSCs). BMSCs were isolated and the cell gender was identified through polymerase chain reaction (PCR). Then different concentrations of 5-aza-CdR (0, 0.02, 0.1 µM) were applied for the treatment. The results showed cellular morphology, growth, Xist gene expression pattern, and adherent ability were not significantly affected with the treatment of 5-aza-CdR for 24 hours. Meanwhile, immunofluorescence analysis indicated that the expression of 5-methylcytosine (5-mC) was also not influenced after the treatment. However, bisulfite sequence PCR (BS-PCR) analysis revealed that the methylation level of Xist differentially methylated region (DMR) decreased significantly when the concentration of 5-aza-CdR increased to 0.1 µM in the ♀BMSCs group (p < 0.05), while there was no significant difference among the ♂BMSCs-treated groups. Our results implied that low concentrations of 5-aza-CdR treatment had little impacts on cellular morphology, growth Xist gene expression pattern, adherent ability, and global DNA methylation level of BMSCs in both genders, but the treatment could significantly decrease the methylation level of Xist DMR in ♀BMSCs. Thus, we conclude 5-aza-CdR treatment can affect the methylation status of Xist DMR, furthermore, the influence is also related to sex differences.

Effect of sex differences in donor foetal fibroblast on the early development and DNA methylation status of buffalo (Bubalus bubalis) nuclear transfer embryos.

Low efficiency of somatic cell nuclear transfer (SCNT) embryos is largely attributable to imperfect reprogramming of the donor nucleus. The differences in epigenetic reprogramming between female and male buffalo cloned embryos remain unclear. We explored the effects of donor cell sex differences on the development of SCNT embryos. We and then compared the expression of DNA methylation (5-methylcytosine-5mC and 5-hydroxymethylcytosine-5hmC) and the expression level of relevant genes, and histone methylation (H3K9me2 and H3K9me3) level in SCNT-♀ and SCNT-♂ preimplantation embryos with in vitro fertilization (IVF) counterparts. In the study, we showed that developmental potential of SCNT-♀ embryos was greater than that of SCNT-♂ embryos (p < 0.05). 5mC was mainly expressed in SCNT-♀ embryos, whereas 5hmC was majorly expressed in SCNT-♂ embryos (p < 0.05). The levels of DNA methylation (5mC and 5hmC), Dnmt3b, TET1 and TET3 in the SCNT-♂ embryos were higher than those of SCNT-♀ embryos (p < 0.05). In addition, there were no significant differences in the expression of H3K9me2 at eight-stage of the IVF, SCNT-♀ and SCNT-♂embryos (p < 0.05). However, H3K9me3 was upregulated in SCNT-♂ embryos at the eight-cell stage (p < 0.05). Thus, KDM4B ectopic expression decreased the level of H3K9me3 and significantly improved the developmental rate of two-cell, eight-cell and blastocysts of SCNT-♂ embryos (p < 0.05). Overall, the lower levels of DNA methylation (5mC and 5hmC) and H3K9me3 may introduce the greater developmental potential in buffalo SCNT-♀ embryos than that of SCNT-♂ embryos.