College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu, People's Republic of China.
Nanjing Agricultural University, Nanjing, Jiangsu, People's Republic of China.
J Biochem Mol Toxicol. 2019 Jul;33(7):e22329. doi: 10.1002/jbt.22329. Epub 2019 Apr 1.
Lipopolysaccharide (LPS) can cause ovarian dysfunction and infertility in mammals. The purpose of this study was to investigate the effects of LPS on the accumulation of lipid droplets (LDs), proliferation, and steroidogenesis in goat luteinized granulosa cells (LGCs). GCs isolated from the ovarian follicles were spontaneously luteinized under media with fetal bovine serum, resulting in increased progesterone and shifted shape from spherical to star with multiple prolongations. Then, LGCs were treated with LPS (0-10 μg/mL) for 0-48 hours. Oil Red O staining was performed to observe LDs accumulation and commercial kit was applied to detect intracellular triglyceride (TG) content. The cell proliferation were detected by cell counting kit-8. Expressions of cell-cycle-related genes were determined by real-time polymerase chain reaction. Estradiol (E ) and progesterone (P ) from cell supernatants were determined by enzyme-linked immunosorbent assay, and expressions of STAR, P450scc, 3β-hydroxysteroid dehydrogenase (3β-HSD) and CYP19A1 were detected by Western blot. Results showed that LPS treatment significantly increased LDs accumulation after 24 hours, and 5 μg/mL LPS increased TG content ( P < 0.05). LPS treatment for 24 hours stimulated the LGCs activities ( P<0.05), which was confirmed by the increases in the expressions of proliferating cell nuclear antigen (PCNA), cyclinB1 and cyclinD1, while 48 hours treatment had no effect. LPS treatment suppressed E and P output of LGCs ( P < 0.05). Western blot results showed that 10 μg/mL LPS decreased the protein expression of 3β-HSD in LGCs ( P < 0.05). In conclusion, LPS increased LDs accumulation and cell proliferation, and LPS-mediated P reduction could be attributed to the decreased 3β-HSD protein expression, which provide new information for the regulation of ovarian function in goats.
脂多糖（LPS）可导致哺乳动物卵巢功能障碍和不孕。本研究旨在探讨 LPS 对山羊黄体化颗粒细胞（LGC）中脂滴（LDs）积累、增殖和类固醇生成的影响。从卵巢卵泡中分离的 GC 在含有胎牛血清的培养基中自发黄体化，导致孕酮增加，细胞形状从球形变为星型，多个突起。然后，用 LPS（0-10μg/ml）处理 LGC 0-48 小时。用油红 O 染色观察 LDs 积累，并用商业试剂盒检测细胞内三酰甘油（TG）含量。通过细胞计数试剂盒-8 检测细胞增殖。通过实时聚合酶链反应测定细胞周期相关基因的表达。通过酶联免疫吸附试验测定细胞上清液中雌二醇（E ）和孕酮（P ）的含量，并用 Western blot 检测 STAR、P450scc、3β-羟甾脱氢酶（3β-HSD）和 CYP19A1 的表达。结果表明，LPS 处理 24 小时后 LDs 积累明显增加，5μg/ml LPS 增加 TG 含量（P<0.05）。LPS 处理 24 小时刺激 LGC 活性（P<0.05），这得到增殖细胞核抗原（PCNA）、细胞周期蛋白 B1 和细胞周期蛋白 D1 表达增加的证实，而 48 小时处理则没有影响。LPS 处理抑制 LGCs 的 E 和 P 分泌（P<0.05）。Western blot 结果表明，10μg/ml LPS 降低了 LGCs 中 3β-HSD 的蛋白表达（P<0.05）。综上所述，LPS 增加了 LDs 的积累和细胞增殖，LPS 介导的 P 减少可能归因于 3β-HSD 蛋白表达的降低，这为调控山羊卵巢功能提供了新的信息。
