The Key Laboratory for Animal Science of National Ethnic Affairs Commission, Southwest Minzu University, Chengdu, 610041, PR China; Zigong Psychiatric Research Center, Zigong, 643020, PR China.
The Key Laboratory for Animal Science of National Ethnic Affairs Commission, Southwest Minzu University, Chengdu, 610041, PR China.
Theriogenology. 2023 Sep 1;207:1-10. doi: 10.1016/j.theriogenology.2023.05.020. Epub 2023 May 23.
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.
血管内皮生长因子（VEGF）通过调节一些哺乳动物的颗粒细胞（GC）功能对卵泡发育至关重要，但在牦牛（Bos grunniens）中的机制尚不清楚。因此，本研究的目的是探讨 VEGF 对牦牛 GC 活力、凋亡和类固醇生成的影响。首先，我们通过免疫组织化学分析研究了 VEGF 及其受体（VEGFR2）在牦牛卵巢中的定位，并通过细胞计数试剂盒-8 评估了含有不同 VEGF 浓度和培养时间的培养基对牦牛 GC 活力的影响。然后，选择 20ng/mL VEGF 培养 24 小时作为最佳处理条件，通过 DCFH-DA 试剂盒分析该化合物对细胞内活性氧水平的影响，通过流式细胞术分析细胞周期和凋亡，通过 ELISA 试剂盒分析类固醇生成，通过 RT-qPCR 分析相关基因的表达。结果表明，VEGF 和 VEGFR2 在 GC 和膜细胞中高度共表达。在含有 20ng/mL VEGF 的培养基中培养 24 小时的 GC 细胞活力显著提高，ROS 产生减少（P<0.05），促进 G1 期向 S 期的转变（P<0.05），CCND1（P<0.05）、CCNE1、CDK2、CDK4 和 PCNA 基因的表达增加（P<0.01），P53 基因的表达减少（P<0.05）。这种治疗通过促进 BCL2 和 GDF9 的表达（P<0.01）和抑制 BAX 和 CASPASE3 的表达（P<0.05），显著降低 GC 凋亡（P<0.05）。VEGF 促进孕酮分泌（P<0.05），同时增加 HSD3B、StAR 和 CYP11A1 的表达（P<0.05）。综上所述，我们的研究结果强调了 VEGF 通过调节相关基因表达对改善 GC 活力、减少 ROS 产生和凋亡率的有益影响。
