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.

Hypoxia promotes steroidogenic competence of buffalo (Bubalus bubalis) theca cells.

Theca cells (TCs) play an important role in follicular development and atresia. TCs synthesize androgens that act as substrate for granulosa cells aromatization to estrogens needed for follicular growth. However, the effects of hypoxia on steroidogenesis in buffalo TCs remain unclear. In the present study, the impacts of hypoxic conditions (5% oxygen) on androgen synthesis in buffalo TCs were examined. The results showed that hypoxia improved both the expression levels of androgen synthesis-related genes (CYP11A1, CYP17A1, and 3ß-HSD) and the secretion levels of testosterone in buffalo TCs. Hypoxic conditions promoted the sensitivity of buffalo TCs to LH. Furthermore, inhibition of PI3K/AKT signaling pathway reduced both the expression levels of androgen synthesis-related genes (CYP11A1, CYP17A1, and 3ß-HSD) and the secretion levels of testosterone in hypoxia-cultured buffalo TCs. Besides, inhibition of PI3K/AKT signaling pathway lowered the sensitivity of buffalo TCs to LH under hypoxic conditions. This study indicated that hypoxia enhanced the steroidogenic competence of buffalo TCs main through activating PI3K/AKT signaling pathway and subsequently facilitating the responsiveness of TCs to LH. This study provides a basis for further exploration of ovarian endocrine mechanism for steroidogenesis.

Granulosa cells affect in vitro maturation and subsequent parthenogenetic development of buffalo (Bubalus bubalis) oocytes.

Granulosa cells (GCs) play a crucial role in follicular development and atresia. Previous studies have showed that GCs in the form of monolayer influenced in vitro maturation (IVM) of oocytes. However, the effects of GCs in the form of conditioned medium and monolayer on IVM and development competence of buffalo oocytes remain unclear. In the present study, we examined the impacts of GC-conditioned medium (GCCM) and monolayer GC on maturation efficiency and embryo development of buffalo oocytes after parthenogenetic activation (PA). Our results showed that GCCM that was collected on day 2 and added to IVM medium at a 20% proportional level (2 days and 20%) exerted significant negative effects on IVM rate (41.6% vs. 44.5%), but significantly enhanced embryo development (oocyte cleavage, 81.3% vs. 69.3%; blastocyst formation, 36.3% vs. 29.3%) of buffalo oocytes after PA compared with the control group. Furthermore, monolayer GC significantly reduced both maturation efficiency (40.2% vs. 44.5%) and embryo development (oocyte cleavage, 60.6% vs. 69.3%; blastocyst formation, 20.6% vs. 29.3%) of buffalo oocytes after PA compared to the control group. Our study indicated that GCs in the form of GCCM (2 days and 20%) and monolayer GC had different effects on IVM and subsequent parthenogenetic development of buffalo oocytes.

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.