人工诱导花鳗鲡成熟过程中卵巢内6种性类固醇激素的含量及变化

Quantification of 6 sexual steroid hormones in the ovary of marbled eel Anguilla marmorata during artificial induced maturation

  • 摘要: 为分析成年花鳗鲡经多次注射鲤鱼脑垂体(carp pituitary extract,CPE,每周12 mg/kg)和人绒毛膜促性腺激素(hCG,每周300 IU/kg)后,卵巢发育成熟过程中6种性类固醇激素的含量及变化,实验采用超高效液相色谱—串联质谱(UPLC/MS/MS)联用方法对花鳗鲡性腺内这6种类固醇激素进行研究。结果发现,睾酮(testosterone,T)、孕酮(progesterone,P)、雌二醇(estradiol-17β,E2)、雌三醇(estriol,E3)、17α,20β-二羟基-4-孕烯-3-酮(17α,20β-dihydroxy-4-pregnen-3-one,DHP)、17α-羟基孕酮(17α-hydroxyprogesterone,17α-OHP)在xBridge C18色谱柱上得到良好分离,在0~200 ng/mL的范围内线性相关系数均大于0.99,检测限为0.1~0.5 ng/g,回收率为89.00%~94.83%,相对标准差均小于20%; 注射外源激素后,6种性类固醇激素在花鳗鲡卵巢中的含量及变化情况如下:第2次注射后,T、P、E2、E3、DHP、17α-OHP的含量分别为(0.27±0.05)、0、(0.64±0.05)、(1.17±0.19)、0、0 ng/g; 第9次注射后,T、P、E2、E3、DHP、17α-OHP的含量分别为(0.73±0.13)、(1.28±0.38)、(1.27±0.27)、(0.83±0.14)、(1.50±0.59)、(0.43±0.25) ng/g; 第16次注射后,T、P、E2、E3、DHP、17α-OHP的含量分别为(1.17±0.14)、(2.23±0.51)、(5.59±0.96)、(2.46±0.70)、(2.29±0.65)、(4.56±0.74) ng/g。对照组中均未检测到雌二醇和雌三醇。研究表明,雌二醇和雌三醇是花鳗鲡性腺发育的关键因素; 随着性腺发育,花鳗鲡卵巢中6种性类固醇激素的含量整体呈逐步增加的趋势。另一方面,UPLC/MS/MS法能同时定量测定这6种性类固醇激素,具有灵敏、高回收率、稳定的优势。

     

    Abstract: To determine the content and changes of 6 steroid hormones in the ovaries of female farmed marbled eels Anguilla marmorata during artificially induced maturation through carp pituitary extract (12 mg/kg·week) and hCG(300 IU/kg·week) injection,in this paper,ultra-performance liquid chromatography coupled with electronspray ionization-triple quadruple mass spectrometry(UPLC/MS/MS) method was applied to detect the 6 steroid hormones..The results showed that the chromatographic separation of 6 gonad steroid hormones,namely,testosterone(T),progesterone(P),estradiol-17β(E2),estriol(E3),17α,20β-dihydroxy-4-pregnen-3- one(DHP),and 17α-hydroxyprogesterone (17α-OHP) was well performed using an xBridge C18 column.The linear regression coefficients(R) of T,P,E2,E3,DHP,17α-OHP were above 0.99 in the range of 0-200 ng/ml and the limits of detection were above 0.1-0.5 ng/g.Average recoveries of T,P,E2,E3,DHP,17α-OHP also reached more than 89.00%-94.83%,and relative standard deviations were less than 20%.The average contents of T,P,E3in the second week(after second injection) were (0.27±0.05),(0.64±0.05),(1.17±0.19)ng/g respectively.DHP,E2,17α-OHP were not detected in the second week.From the ninth week,the contents of T,P,E2,E3 DHP,17α-OHP increased to (0.73±0.13),(1.28±0.270.38),(1.27±0.27),(0.83±0.14),(1.50±0.59),(1.43±0.25)ng/g.The average contents of T,P,E2,E3,DHP,17α-OHP in sixteenth week (after sixteenth injection) were (1.17±0.14),(2.23±0.51),(5.59±0.96),(2.46±0.70),(2.29±0.65),(4.56±0.74)ng/g,while there were no E2 and E3 detected in the control group.The results indicated that E2 and E3 are essential to ovary development,and the 6 steroid hormones increased with the development of gonad maturation.The UPLC/MS/MS method is a sensitive and reliable approach with high recovery rate in simultaneously quantifying 6 gonad steroid hormones in the fish.

     

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