生殖激素对山羊颗粒细胞孕酮合成的影响  

Effects of reproductive hormones on progesterone synthesis in granulosa cells of goats

作　　者：李曼曼 于昊 薛洋 茆达干[1,2] LI Man-man;YU Hao;XUE Yang;MAO Da-gan(College of Animal Science and Technolo-gy.Nanjing Agricultural University,Nanjing 210095,China;National Ex perimental Teaching Demonstration Center of Animal Science,Nanjing Agricultural Unirversity,Nanjing 210095,Chi-na)

机构地区：[1]南京农业大学动物科技学院,江苏南京210095 [2]南京农业大学动物科学类国家级实验教学示范中心,江苏南京210095

出　　处：《中国兽医学报》2020年第8期1652-1659,共8页Chinese Journal of Veterinary Science

基　　金：国家重点研发计划资助项目(2018YFD0501906);江苏省重点研发计划(现代农业)资助项目(BE2017320)。

摘　　要：试验以山羊颗粒细胞为研究对象,采集卵泡(φ2~5 mm)颗粒细胞进行无血清培养2 d后,用FSH、LH(2.5 IU/mL或1.25 IU/mL)和E2(1 mg/L)单独或联合处理0~96 h,分别收集细胞和培养液,用放射免疫分析法(RIA)检测培养液中孕酮(P4)水平,用实时荧光定量PCR(qPCR)和蛋白免疫印迹(WB)技术检测颗粒细胞孕酮相关基因和蛋白的表达。结果显示,激素联合或单独处理24 h后,FSH/LH/E2(2.5/2.5/1)组的孕酮水平与LH(2.5)组相当,均显著高于其他4组(P<0.01),然而,在处理48,72,96 h时间点,各组间均无显著性差异(P>0.05)。FSH/LH/E2(2.5/2.5/1)组处理后72,96 h的孕酮水平极显著低于0,24 h(P<0.01);FSH/LH/E2(1.25/1.25/1)组与FSH(2.5)组的孕酮水平24 h显著下降(P<0.05),但48 h又显著高于24 h(P<0.05);LH(2.5)组的孕酮水平96 h显著下降(P<0.05);E2(1)组和对照组的孕酮水平均在24 h显著下降(P<0.05)。qPCR结果显示,FSH/LH/E2(2.5/2.5/1)处理不同时间后PTGFR、HSD3B和StAR基因在24 h表达量极显著升高(P<0.01),随后表达量均逐渐下降,且处理后96 h的基因表达量均极显著低于0 h组(P<0.01)。WB结果显示,激素处理24 h后HSD3B在FSH/LH/E2(2.5/2.5/1)组的蛋白表达量极显著高于其他组(P<0.01);P450scc在FSH/LH/E2(2.5/2.5/1)组的蛋白表达量与对照组相比有升高趋势(P=0.0604),其他各组间无趋势变化(P>0.1);而CYP19A1表达量在6个组中无显著性差异(P>0.05)。结果表明,FSH、LH和E2联合处理更有利于体外无血清培养的山羊颗粒细胞合成孕酮,为进一步研究生殖激素在山羊卵巢卵泡-黄体转化中的作用奠定理论基础。Goat granulosa cells(GCs)were isolated from healthy follicles(φ2~5 mm)and cultured in complete culture media for two days.Then,FSH(2.5 IU/mL or 1.25 IU/mL),LH(2.5 IU/mL or 1.25 IU/mL)and E2(1 mg/L)were added for further culture and cell supernatant was collected at different time points(0,24,48,72 and 96 h)to detect P4 level by RIA.Cells were collected to detect the expression of progesterone related genes and proteins by quantitative real-time PCR(qPCR)and Western blot(WB).The results showed that the progesterone level in FSH/LH/E2(2.5/2.5/1)group,similar to that in LH(2.5)group,was significantly higher than that in other groups after 24 h(P<0.01).However,there was no significant difference among all the groups at the time points of 48,72 and 96 h(P>0.05).In the FSH/LH/E2(2.5/2.5/1)group,the progesterone levels at 72 h and 96 h were significantly lower than those at 0 h and 24 h(P<0.01).The progesterone levels in the FSH/LH/E2(1.25/1.25/1)group and the FSH(2.5)group showed the same trend,both significantly decreased at 24 h(P<0.05),and then increased significantly at 48 h(P<0.05).The progesterone level in the LH(2.5)group significantly decreased at 96 h(P<0.05).The progesterone level in both E2(1)group and control group decreased significantly at 24 h(P<0.05).qPCR results showed that in the FSH/LH/E2(2.5/2.5/1)group,the expression levels of PTGFR,HSD3 Band StAR increased significantly at 24 hafter treatment(P<0.01),and then decreased gradually,significantly lower at 96 hthan those at 0 h(P<0.01).WB results showed that after 24 htreatment,the HSD3 Bexpression in FSH/LH/E2(2.5/2.5/1)group was significantly higher than that in other groups(P<0.01).There was an increased tendency in P450 scc expression in FSH/LH/E2(2.5/2.5/1)group compared with the control group(P=0.0604),and no significant difference in CYP19 A1 expression among all the six groups(P>0.05).Combined treatment with FSH,LH and E2 could promote the synthesis of progesterone in goat granulosa cells cultured without serumin vitro,which lay a theoretical

关 键 词：生殖激素 山羊 颗粒细胞 无血清培养 P4 

分 类 号：S813[农业科学—畜牧学] S852.2[农业科学—畜牧兽医]