Evidence for two-cell model of steroidogenesis in four species of amphibian

Previously, based on studies conducted using Rana nigromaculata, a two-cell model involving the theca and granulosa cells was proposed to account for the steroidogenic activity of amphibian ovarian follicles. Experiments were carried out to ascertain whether the model was applicable to four other fr og species with different reproductive cycles (R. dybowskii, R. rugosa, R. catesbeiana, and Bombina orientalis). Ovarian follicles were collected from each species and manually microdissected to obtain various follicular comp onents: theca-epithelium (THEP) and granulosa cell-enclosed oocyte (GCEO). Subsequent to collection, equal numbers of intact follicles and various fol licular components were cultured for 6 hr in the presence of known inducers of steroidogenesis (frog pituitary homogenate [FPH] or 3-iso-butyl-1-methy lxanthine [IBMX] + forskolin) or various steroids that serve as substrates for specific steroidogenic enzymes. Following incubation, culture medium wa s collected and analyzed by radioimmunoassay (RIA). Both FPH and IBMX + for skolin consistently stimulated secretion of androstenedione (AD), testoster one (T), and estradiol (E-2) from intact follicles obtained from all four f rog species. Additionally, in R. dybowskii, these treatments stimulated sec retion of progesterone (P-4) and 17 alpha-hydroxyprogesterone (17 alpha-OHP 4) into the culture medium. Intact follicles obtained from all species read ily converted pregnenolone (P-5), P-4, and 17 alpha-OHP4 to AD, T, and E-2. In contrast GCEO converted P-5, P-4, and 17 alpha-OHP4 to AD and E-2, but not to T. However, AD, but not P-5, P-4, or 17 alpha-OHP4, was converted to T when cultured in the presence of isolated THEP. The microdissection proc edure was also modified to isolate THEP without contaminating granulosa cel ls. The steroidogenic capacities of "impure" THEP and "pure" theca-epitheli um (P-THEP) were then compared. Basal amounts of Pq Were produced when Pg w as added to P-THEP, whereas significantly higher amounts were produced in t he presence of impure THEP. No significant conversion of P-5 or P-4 to 17 a lpha-OHP4 occurred following culture with pure or impure THEP layer. Result s suggest that the enzyme activity necessary to metabolize AD --> T is loca lized in the THEP, whereas the metabolic capacities to convert P-5 --> AD a nd T --> E-2 are present in the granulosa cell. Furthermore, the data show that the two-cell model is applicable to other frog species. J. Exp. Zool. 284:91-99, 1999. (C) 1999 Wiley-Liss, Inc.