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Abstract：
Energy and the cAMP-response element binding protein (CREB)/steroidogenic acute regulatory protein (StAR) signaling pathway play important roles in steroid hormone production and follicular development in hens. This present study aimed to investigate the effects of exogenous energy on the synthesis of steroid hormones and the expression characteristics of the CREB/StAR signaling pathway in theca cells of laying hen. The primary theca cells of small yellow follicles were randomly divided into 6 treatments and cultured in medium with glucose concentrations of 1, 1.5, 3, 4.5, 6, and 7.5 mg/mL for 48 h. It was found that growth was robust and cell outlines were clear when cells were cultured with 1, 1.5, 3, and 4.5 mg/mL glucose, but cell viability was diminished and cell density decreased after exposure to glucose at 6 and 7.5 mg/mL for 48 h. Cell viability showed an increasing and then decreasing quadratic response to increasing glucose concentration in culture (r2 = 0.688, P < 0.001). The cell viability of theca cells cultured with 4.5 mg/mL glucose was greater than those cultured with 1, 1.5, 6, and 7.5 mg/mL glucose (P < 0.05). The concentration of estradiol in the medium containing 3 mg/mL glucose was higher than in medium containing 1, 1.5, and 6 mg/mL glucose (P < 0.05). There was an increasing and then decreasing quadratic correlation between progesterone concentrations and glucose concentrations (r2 = 0.522, P = 0.002). The concentration of progesterone in medium with 4.5 mg/mL glucose was higher than in medium with 1 and 7.5 mg/mL glucose (P < 0.05). There was an increasing and then decreasing quadratic correlation between the relative expression of CREB1 (r2 = 0.752, P < 0.001), StAR (r2 = 0.456, P = 0.002), CYP1B1 (r2 = 0.568, P < 0.001), and 3β-HSD (r2 = 0.319, P = 0.018) in theca cells of laying hens and glucose concentrations after treatment with different glucose concentrations for 48 h. After treatment with 4.5 mg/mL glucose, the expression of StAR, CYP1B1, and 3β-HSD genes were increased compared to treatment with 1, 1.5, 3, 6, and 7.5 mg/mL glucose (P < 0.001). There was an increasing and then decreasing quadratic correlation between glucose concentrations and protein expression of CREB1 (r2 = 0.819, P < 0.001), StAR (r2 = 0.844, P < 0.001), 3β-HSD (r2 = 0.801, P < 0.001), and CYP11A1 (r2 = 0.800, P < 0.001) in theca cells of laying hens. The protein expression of CREB1, StAR, and 3β-HSD in theca cells cultured with 4.5 mg/mL glucose was higher than in other groups (P < 0.001). The results indicate that the appropriate glucose concentration (4.5 mg/mL) can improve the synthesis of steroid hormones in theca cells of laying hens through the upregulation of key genes and proteins in the CREB/StAR signaling pathway.
摘要：
能量和cAMP反应元件结合蛋白（CREB）/类固醇生成急性调节蛋白（StAR）信号通路在母鸡类固醇激素产生和卵泡发育中起重要作用。本研究旨在探讨外源能量对蛋鸡卵泡膜细胞类固醇激素合成及CREB/StAR信号通路表达特征的影响。将黄色小卵泡的原代卵泡膜细胞随机分为6个处理，在葡萄糖浓度为1、1.5、3、4.5、6和7.5mg/mL的培养基中培养48h。发现当细胞分别用1、1.5、3和4.5mg/mL葡萄糖培养时，生长强劲，细胞轮廓清晰，但暴露于6和7.5mg/mL的葡萄糖48h后，细胞活力降低，细胞密度降低。细胞活力对培养物中葡萄糖浓度的增加表现出增加然后降低的二次反应（r2=0.688，P<0.001）。用4.5mg/mL葡萄糖培养的卵泡膜细胞的细胞活力高于用1、1.5、6和7.5mg/mL葡萄糖培养的细胞(P<0.05)。含3mg/mL葡萄糖的培养基中雌二醇的浓度高于含1、1.5和6mg/mL葡萄糖的培养基（P<0.05）。孕酮浓度与葡萄糖浓度之间存在先增加后减少的二次相关性（r2=0.522，P=0.002）。4.5mg/mL葡萄糖培养基中的孕酮浓度高于1和7.5mg/mL葡萄糖培养基中的孕酮浓度（P<0.05）。CREB1的相对表达之间存在先升高后降低的二次相关性(r2=0.752,P<0.001),StAR(r2=0.456,P=0.002),CYP1B1(r2=0.568,P<0.001),用不同葡萄糖浓度处理48h后，蛋鸡卵泡膜细胞中的3β-HSD（r2=0.319，P=0.018）和葡萄糖浓度。用4.5mg/mL葡萄糖处理后，StAR的表达,与1、1.5、3、6和7.5mg/mL葡萄糖治疗相比，CYP1B1和3β-HSD基因增加（P<0.001）。葡萄糖浓度与CREB1蛋白表达呈先递增后递减的二次相关关系(r2=0.819,P<0.001),StAR(r2=0.844,P<0.001),3β-HSD（r2=0.801，P<0.001），产蛋鸡卵泡膜细胞中的CYP11A1（r2=0.800，P<0.001）。CREB1、StAR、4.5mg/mL葡萄糖培养的卵泡膜细胞中3β-HSD高于其他组（P<0.001）。结果表明,适宜的葡萄糖浓度(4.5mg/mL)可通过上调CREB/StAR信号通路中的关键基因和蛋白,促进蛋鸡卵泡膜细胞类固醇激素的合成。
