Table_1_β-NGF Stimulates Steroidogenic Enzyme and VEGFA Gene Expression, and Progesterone Secretion via ERK 1/2 Pathway in Primary Culture of Llama Granulosa Cells.docx

The beta-nerve growth factor (β-NGF) from llama seminal plasma exerts ovulatory and luteotrophic effects following intramuscular or intrauterine infusion in llamas and alpacas. In this study, we investigate the in vitro effect of llama β-NGF on the expression of genes involved in angiogenesis and progesterone synthesis as well as progesterone release in preovulatory llama granulosa cells; we also determine whether these changes are mediated via the ERK1/2 signaling pathway. From adult female llamas, we collected granulosa cells from preovulatory follicles by transvaginal ultrasound-guided follicle aspiration; these cells were pooled and incubated. After 80% confluence, the cultured granulosa cells were treated with β-NGF, β-NGF plus the MAPK inhibitor U0126, or luteinizing hormone, and the abundance of angiogenic and steroidogenic enzyme mRNA transcripts were quantified after 10 and 20 h by RT-qPCR. We also quantified the progesterone concentration in the media after 48 h by radioimmunoassay. We found that application of β-NGF increases the abundance of mRNA transcripts of the vascular endothelial growth factor (VEGFA) and the steroidogenic enzymes cytochrome P450 side-chain cleavage (P450scc/CYP11A1), steroidogenic acute regulatory protein (STAR), and 3β-hydroxysteroid dehydrogenase (HSD3B1) at 10 and 20 h of treatment. Application of the MAPK inhibitor U0126 resulted in downregulation of the genes encoding these enzymes. β-NGF also enhanced progesterone synthesis, which was prevented by the prior application of the MAPK inhibitor U0126. Finally, western blot analysis confirmed that β-NGF activates the ERK1/2 signaling pathway. In conclusion, our results indicate that β-NGF exerts direct luteotropic effects on llama ovarian tissue via the ERK 1/2 pathway.

来自大羊驼精浆的β-神经生长因子（beta-nerve growth factor，β-NGF）在大羊驼和羊驼中经肌肉内或子宫内输注后，可发挥排卵及黄体营养作用。本研究旨在探究大羊驼来源的β-NGF对排卵前大羊驼颗粒细胞中血管生成、孕酮合成相关基因表达及孕酮释放的体外影响，并明确上述变化是否通过ERK1/2信号通路介导。本研究通过经阴道超声引导下卵泡抽吸术，从成年雌性大羊驼的排卵前卵泡中采集颗粒细胞，将收集的细胞混合后进行培养。当培养的颗粒细胞汇合度达80%时，分别用β-NGF、β-NGF联合丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）抑制剂U0126以及促黄体生成素（luteinizing hormone，LH）进行处理，并于处理后10 h和20 h通过实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）定量检测血管生成酶与类固醇生成酶的mRNA转录本丰度。本研究同时于处理后48 h通过放射免疫分析法（radioimmunoassay，RIA）定量检测培养基中的孕酮浓度。研究结果显示，在处理后10 h和20 h，β-NGF处理可上调血管内皮生长因子（vascular endothelial growth factor，VEGFA）以及类固醇生成酶细胞色素P450侧链裂解酶（cytochrome P450 side-chain cleavage，P450scc/CYP11A1）、类固醇生成急性调节蛋白（steroidogenic acute regulatory protein，STAR）和3β-羟类固醇脱氢酶（3β-hydroxysteroid dehydrogenase，HSD3B1）的mRNA转录本丰度；联合使用丝裂原活化蛋白激酶抑制剂U0126则可下调上述酶编码基因的表达。β-NGF还可促进孕酮合成，而预先使用丝裂原活化蛋白激酶抑制剂U0126可阻断这一效应。最后，蛋白质免疫印迹（western blot）分析证实，β-NGF可激活ERK1/2信号通路。综上，本研究结果表明，β-NGF可通过ERK1/2通路对大羊驼卵巢组织发挥直接的黄体营养作用。