Characterization of parameters for in vitro culture of isolated ovarian follicles of greenback flounder Rhombosolea tapirina

Isolated ovarian follicles of greenback flounder Rhombosolea tapirina were incubated with a variety of gonadotropins (GtHs) and steroid precursors for periods of up to 42 h, and levels of free and glucuronated testosterone (T) and 17β-estradiol (E2) in the medium, and free T and E2 from inside follicles were measured by RIA. Short incubations (6 h) generated increases in T and E2 in response to steroid precursors, but not human chorionic GtH (hCG), or salmon or carp GtH. At incubation times of 18 h, all GtHs stimulated T and, or E2 production, whereas after 42-h incubation, GtH effects on E2 production had disappeared. Steroid precursors remained effective at 18 and 42 h. T and E2 glucuronides were formed in small quantities but did not account for loss of treatment effects at long incubation times. Instead, this could be explained by accumulation of E2 in controls as a result of continued basal steroid production. Follicles absorbed substantial amounts of both endogenous and exogenous steroid from the medium, however, this did not appear to have any influence on changes in treatment effects with incubation time. Flounder follicles were most sensitive to hCG, followed by salmon and carp GtH at approximately 10-fold higher concentrations. Ovarian segments were not sensitive to any GtH but did convert exogenous steroid precursors indicating that tissue access by GtH may be a limiting factor under certain in vitro conditions. HCG augmented the conversion of 17-hydroxyprogesterone (17P) to T but not T to E2, consistent with the relative GtH-insensitivity of aromatase in other species. Follicles converted a range of steroid precursors with equal competence, indicating that no step in the cleavage pathway is strongly rate-limited, and that choice of precursor is unlikely to affect the assessment of steroidogenic activity. (C) 2000 Elsevier Science Inc.