摘要
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.
摘要译文
在体外成熟过程中补充乙酰基-L-肉碱（ALC）可显着提高卵母细胞裂解的速率，羊毛菌和绵羊和水牛中的胚泡形成；但是，ALC在提高卵母细胞能力方面的作用方式尚不完全了解。因此，这项研究的目的是研究ALC对YAK（BOS Grunniens）颗粒细胞（GCS）中抗氧化特性，脂质液滴积累和类固醇激素分泌的影响。使用FSHR免疫荧光鉴定YAK GC。用不同浓度的ALC处理细胞，通过细胞计数KIT-8检测细胞增殖，并确定最佳浓度和治疗时间以进行随后的实验。然后，通过DCFH-DA探针检测到活性氧（ROS），并通过油红O染色观察到脂质液滴积累。通过ELISA检测到培养基中的雌二醇（E2）和孕酮（P4），并通过RT = QPCR确定与细胞增殖，凋亡，细胞周期，抗氧化剂和类固醇合成有关的基因的表达。结果表明，1 mM ALC治疗48小时是最佳治疗方法。它显着提高了细胞活力（P <0.05），显着降低了ROS和脂质液滴含量的量，并促进了YAK GC的P4和E2分泌（P <0.05）。RT - QPCR结果证实了用1 mM ALC处理48小时的GCS显着增加了与抗凋亡和细胞周期相关的基因表达（Bcl-2，PCNA，CCND1和CCNB1），抗氧化剂（CAT，SOD2和GPX1），以及E2和P4分泌（Star，CYP19A1和HSD3B1）（P <0.05），但它显着降低了凋亡基因的表达（Bax和P53）（P <0.05）。总之，ALC提高了YAK GC的活力，减少了ROS和脂质液滴的量，增加了P4和E2合成，并影响了YAK GC中相关基因的表达。
Xu-dong Jiang; Yu Liu; Jian-fei Wu; San-ni Gong; ... Xiang-dong Zi. Regulation of proliferation, apoptosis, hormone secretion and gene expression by acetyl-L-carnitine in yak (Bos grunniens) granulosa cells[J]. Theriogenology, 2023,203: 61-68