BACKGROUND Nerve growth factor (β-NGF) from llama seminal plasma has been described as a potent ovulatory and luteotrophic molecule after intramuscular or intrauterine infusion in llamas and alpacas. We tested the hypothesis that systemic administration of purified β-Nerve Growth Factor (β-NGF) during the preovulatory stage will up-regulate steroidogenic enzymes and Vascular Endothelial Growth Factor (VEGF) gene expression in granulosa cells inducing a change in the progesterone/estradiol ratio in the follicular fluid in llamas. METHODS Experiment I: Female llamas (n = 64) were randomly assigned to receive an intramuscular administration of: a) 50 μg gonadorelin acetate (GnRH, Ovalyse, Pfizer Chile SA, Santiago, Chile, n = 16), b) 1.0 mg of purified llama β-NGF (n = 16), or c) 1 ml phosphate buffered saline (PBS, negative control group, n = 16). An additional group of llamas (n = 16) were mated with a fertile male. Follicular fluid and granulosa cells were collected from the preovulatory follicle at 10 or 20 h after treatment (Time 0 = administration of treatment, n = 8/treatment/time point) to determine progesterone/estradiol concentration and steroidogenic enzymes and VEGF gene expression at both time points. Experiment II: Granulosa cells were collected from preovulatory follicles from llamas (n = 24) using ultrasound-guided transvaginal follicle aspiration for in vitro culture to determine mRNA relative expression of Steroidogenic Acute Regulatory Protein (StAR) and VEGF at 10 or 20 h (n = 4 replicates) and progesterone secretion at 48 h (n = 4 replicates) after LH or β-NGF treatment. RESULTS Experiment I: There was a significant increase in the progesterone/estradiol ratio in mated llamas or treated with GnRH or purified β-NGF. There was a significant downregulation in the mRNA expression of Aromatase (CYP19A1/P450 Arom) for both time points in llamas mated or treated with GnRH or llama purified β-NGF with respect to the control group. All treatments except β-NGF (20 h) significantly up regulated the mRNA expression of 3-beta-hydroxysteroid dehydrogenase (HSD3B) whereas the expression of StAR and Side-Chain cleavage enzyme (CYP11A1/P450scc) where significantly up regulated only by mating (20 h), or β-NGF at 10 or 20 h after treatment. VEGF was up regulated only in those llamas submitted to mating (10 h) or treated with purified β-NGF (10 and 20 h). Experiment II: Only β-NGF treatment induced an increase of mRNA abundance of StAR from llama granulosa cells at 20 h of in vitro culture. There was a significant increase on mRNA abundance of VEGF at 10 and 20 h of in vitro culture from granulosa cells treated with β-NGF whereas LH treatment increases VEGF mRNA abundance only at 20 h of in vitro culture. In addition, there was a significant increase on progesterone secretion from llama granulosa cells 48 h after LH or β-NGF treatment. CONCLUSIONS Systemic administration of purified β-NGF from llama seminal fluid induced a rapid shift from estradiol to progesterone production in the preovulatory follicle. Differences in gene expression patterns of steroidogenic enzymes between GnRH and mated or β-NGF-treated llamas suggest local effects of seminal components on the preovulatory follicle.

中文翻译：

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精浆β-NGF对美洲驼颗粒细胞中卵泡液激素浓度和类固醇生成酶基因表达的影响。

背景技术美洲驼精浆中的神经生长因子（β-NGF）被描述为在美洲驼和羊驼中进行肌肉内或子宫内输注后的有效排卵和黄体营养分子。我们测试了以下假设：在排卵前阶段全身施用纯化的β-神经生长因子（β-NGF）将上调颗粒细胞中的类固醇生成酶和血管内皮生长因子（VEGF）基因表达，从而引起孕酮/雌二醇的变化。骆马卵泡液中的比率。方法实验I：随机分配雌性美洲驼（n = 64）接受以下肌肉注射：a）50μg醋酸促性腺激素（GnRH，Ovalyse，Pfizer Chile SA，Santiago，Chile，n = 16），b）1.0 mg纯化的美洲驼β-NGF（n = 16），或c）1 ml磷酸盐缓冲液（PBS，阴性对照组，n = 16）。另一组美洲驼（n = 16）与一个可育的雄性交配。在治疗后10或20小时（排卵时间0 =给药，n = 8 /治疗/时间点），从排卵前卵泡中收集卵泡液和颗粒细胞，以确定孕激素/雌二醇浓度和类固醇生成酶以及两者的VEGF基因表达时间点。实验II：使用超声引导的经阴道卵泡抽吸法从美洲驼（n = 24）的排卵前卵泡中收集颗粒细胞进行体外培养，以确定在10或20 h（n）的类固醇急性调节蛋白（StAR）和VEGF的mRNA相对表达LH或β-NGF治疗后48小时（n = 4个重复）重复4次重复）和孕激素分泌。结果实验一：在交配的美洲驼中或经GnRH或纯化的β-NGF处理的孕酮/雌二醇比有显着增加。相对于对照组，在用GnRH或美洲驼纯化的β-NGF交配或处理的美洲驼中，两个时间点的芳香酶（CYP19A1 / P450 Arom）的mRNA表达均显着下调。除β-NGF以外的所有处理（20小时）均显着上调了3-β-羟基类固醇脱氢酶（HSD3B）的mRNA表达，而StAR和侧链裂解酶（CYP11A1 / P450scc）的表达仅通过交配显着上调（ 20小时），或在治疗后10或20小时使用β-NGF。VEGF仅在交配（10小时）或用纯化的β-NGF处理（10和20小时）的那些美洲驼中被上调。实验二：在体外培养20 h时，只有β-NGF处理诱导了美洲驼颗粒细胞StAR的mRNA丰度增加。β-NGF处理的颗粒细胞在体外培养10和20 h时，VEGF mRNA的丰度显着增加，而LH处理仅在体外培养20 h时才增加VEGF mRNA的丰度。另外，LH或β-NGF处理48小时后，美洲驼颗粒细胞的孕酮分泌显着增加。结论从美洲驼精液中全身注射纯化的β-NGF，可导致排卵前卵泡中雌二醇的生成迅速转变为孕酮的生成。GnRH与交配或经β-NGF处理的美洲驼之间类固醇生成酶基因表达模式的差异表明精液成分对排卵前卵泡的局部作用。