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 17 beta -estradiol (E-2) in the medium, and free T and E-2 from insid e follicles were measured by RIA. Short incubations (6 h) generated increas es in T and E-2 in response to steroid precursors; but not human chorionic GtH (hCG), or salmon or carp GtH. At incubation times of 18 h, all GtHs sti mulated T and, or E-2 production, whereas after 42-h incubation, GtH effect s on E-2 production had disappeared. Steroid precursors remained effective at 18 and 42 h. T and E-2 glucuronides were formed in small quantities but did not account for loss of treatment effects at long incubation times. Ins tead, this could be explained by accumulation of E-2 in controls as a resul t of continued basal steroid production. Follicles absorbed substantial amo unts of both endogenous and exogenous steroid from the medium, however, thi s did not appear to have any influence on changes in treatment effects with incubation time. Flounder follicles were most sensitive to hCG, followed b y salmon and carp GtH at approximately 10-fold higher concentrations. Ovari an segments were not sensitive to any GtH but did convert exogenous steroid precursors indicating that tissue access by GtH may be a limiting factor u nder certain in vitro conditions. HCG augmented the conversion of 17-hydrox yprogesterone (17P) to T but not T to E-2, consistent with the relative GtH -insensitivity of aromatase in other species. Follicles converted a range o f 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 Elsev ier Science Inc. All rights reserved.