Resveratrol Promotes Proliferation, Antioxidant Properties, and Progesterone Production in Yak (Bos grunniens) Granulosa Cells.

Resveratrol (RES) is a class of natural polyphenolic compounds known for its strong anti-apoptotic and antioxidant properties. Granulosa cells (GCs) are one of the important components of ovarian follicles and play crucial roles in follicular development of follicles in the ovary. Here, we explored the effects of RES on the proliferation and functions of yak GCs. Firstly, we evaluated the effect of RES dose and time in culture on the viability of GCs, and then the optimum treatment protocol (10 µM RES, 36 h) was selected to analyze the effects of RES on the proliferation, cell cycle, apoptosis, malondialdehyde (MDA), glutathione (GSH), reactive oxygen species (ROS) accumulation, lipid droplet content, ATP production, and steroidogenesis of GCs, as well as the expression of related genes. The results show that RES treatment significantly (1) increased cell viability and proliferation and inhibited cell apoptosis by upregulating BCL-2 and SIRT1 genes and downregulating BAX, CASP3, P53, and KU70 genes; (2) increased the proportion of GCs in the S phase and upregulated CCND1, PCNA, CDK4, and CDK5 genes; (3) reduced ROS accumulation and MDA content and increased GSH content, as well as upregulating the relative expression levels of CAT, SOD2, and GPX1 genes; (4) decreased lipid droplet content and increased ATP production; (5) promoted progesterone (P4) secretion and the expression of P4 synthesis-related genes (StAR, HSD3B1, and CYP11A1); and (6) inhibited E2 secretion and CYP19A1 expression. These findings suggest that RES at 10 µM increases the proliferation and antioxidant properties, inhibits apoptosis, and promotes ATP production, lipid droplet consumption, and P4 secretion of yak GCs.

Effects of vascular endothelial growth factor (VEGF) on the viability, apoptosis and steroidogenesis of yak (Bos grunniens) granulosa cells.

Vascular endothelial growth factor (VEGF) is crucial for follicle development through the regulation of granulosa cell (GC) function in some mammals, but its mechanism is unclear in yak (Bos grunniens). Therefore, the objectives of this study were to investigate the effects of VEGF on the viability, apoptosis and steroidogenesis of yak GCs. First, we investigated the localization of VEGF and its receptor (VEGFR2) in yak ovaries by immunohistochemistry analysis and evaluated the effect of culture medium containing different VEGF concentrations and culture times on the viability of yak GCs by Cell Counting Kit-8. Then, optimal treatment with 20 ng/mL VEGF for 24 h was selected to analyze the effects of this compound on intracellular reactive oxygen species levels by DCFH-DA kit, cell cycle and apoptosis by flow cytometry, steroidogenesis by ELISA kit and the expression of the related genes by RT‒qPCR. The results showed that VEGF and VEGFR2 were highly coexpressed in GCs and theca cells. GCs cultured in medium containing 20 ng/mL VEGF for 24 h significantly improved cell viability, decreased ROS production, promoted the transition from G1 phase to S phase (P < 0.05), increased the expression of the CCND1 (P < 0.05), CCNE1, CDK2, CDK4, and PCNA genes (P < 0.01) and decreased the expression of the P53 gene (P < 0.05). This treatment significantly reduced GC apoptosis (P < 0.05) by promoting the expression of BCL2 and GDF9 (P < 0.01) and inhibiting the expression of BAX and CASPASE3 (P < 0.05). VEGF promoted progesterone secretion (P < 0.05) accompanied by increased expression of HSD3B, StAR and CYP11A1 (P < 0.05). Taken together, our findings highlight the beneficial influence exerted by VEGF in improving GC viability and reducing ROS production and the apoptosis rate through the modulation of related gene expression.

Regulation of proliferation, apoptosis, hormone secretion and gene expression by acetyl-L-carnitine in yak (Bos grunniens) granulosa cells.

Supplementation with acetyl-l-carnitine (ALC) during in vitro maturation significantly improves the rates of oocyte cleavage and morula and blastocyst formation in sheep and buffalo; however, the mode of action of ALC in improving oocyte competence is not completely understood. Therefore, the aim of this study was to investigate the effects of ALC on proliferation, antioxidant properties, lipid droplet accumulation and steroid hormone secretion in yak (Bos grunniens) granulosa cells (GCs). Yak GCs were identified using FSHR immunofluorescence. The cells were treated with different concentrations of ALC, cell proliferation was detected by cell counting kit-8, and the optimal concentration and treatment time were determined for subsequent experiments. Then, reactive oxygen species (ROS) were detected by a DCFH-DA probe, and lipid droplet accumulation was observed by oil red O staining. Estradiol (E2) and progesterone (P4) in the medium were detected by ELISA, and the expression of genes related to cell proliferation, apoptosis, the cell cycle, antioxidants and steroid synthesis was determined by RT‒qPCR. The results showed that 1 mM ALC treatment for 48 h was the optimum treatment. It significantly increased cell viability (P < 0.05), significantly decreased the amount of ROS and lipid droplet content, and promoted P4 and E2 secretion (P < 0.05) of yak GCs. RT‒qPCR results verified that GCs treated with 1 mM ALC for 48 h significantly increased the expression of genes related to anti-apoptosis and the cell cycle (BCL-2, PCNA, CCND1 and CCNB1), antioxidants (CAT, SOD2 and GPX1), and E2 and P4 secretion (StAR, CYP19A1 and HSD3B1) (P < 0.05), but it significantly decreased the expression of apoptosis genes (BAX and P53) (P < 0.05). In conclusion, ALC increased the viability of yak GCs, reduced the amount of ROS and lipid droplets, increased P4 and E2 synthesis and affected the expression of related genes in yak GCs.