HPLC-electrochemical detection of ovarian estradiol-17beta and catecholestrogens in the catfish Heteropneustes fossilis: seasonal and periovulatory changes.

A high performance liquid chromatography-electrochemical (HPLC-EC) detection method was used to characterize estradiol-17beta (E2) and its metabolites (2-hydroxyE2, 4-hydroxyE2, and 2-methoxyE2) and investigate their seasonal and periovulatory changes in the ovary of the catfish Heteropneustes fossilis. The retention times in minutes of standards determined by individual and mixture applications are: 2-OHE2-6.6, 4-OHE2-7.0, 4-OHE1-11.2, E2-12.0, and 2-methoxyE2-15.2. Since the retention times of 2-OHE2 and 4-OHE2 merged at higher concentrations, the elution peaks of the sample were taken as due to both (2/4-OHE2) for analysis. The steroids were not detectable in the resting and postspawning phases and 2-methoxyE2 was not detectable in the recrudescent (preparatory, prespawning, and spawning) phases as well. E2 and 2/4-OHE2 have maintained an inverse relationship in the recrudescent phase. The E2 concentration was the highest in the preparatory phase (April) with active vitellogenic activity and declined significantly across prespawning and spawning phases (P<0.001, one way ANOVA; P<0.05, Newman-Keuls' test). On the other hand, the concentration of 2/4-OHE2, which was the lowest in the preparatory phase, increased significantly to the peak level in the spawning phase. A single intraperitoneal injection of hCG (100 IU/fish) stimulated significantly the formation of 2/4-OHE2 at 8 h with a simultaneous reduction in E2. 2-MethoxyE2 was detected only after 16 h of the hCG injection. The functional significance of catecholestrogens in the seasonal reproductive cycle and during the hCG-induced ovulation of the catfish was discussed.