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 frog 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 components: theca-epithelium (THEP) and granulosa cell-enclosed oocyte (GCEO). Subsequent to collection, equal numbers of intact follicles and various follicular components were cultured for 6 hr in the presence of known inducers of steroidogenesis (frog pituitary homogenate [FPH] or 3-iso-butyl-1-methylxanthine [IBMX] + forskolin) or various steroids that serve as substrates for specific steroidogenic enzymes. Following incubation, culture medium was collected and analyzed by radioimmunoassay (RIA). Both FPH and IBMX + forskolin consistently stimulated secretion of androstenedione (AD), testosterone (T), and estradiol (E-2) from intact follicles obtained from all four frog species. Additionally, in R. dybowskii, these treatments stimulated secretion of progesterone (P-4) and 17 alpha-hydroxyprogesterone (17 alpha-OHP4) into the culture medium. Intact follicles obtained from all species readily 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 procedure was also modified to isolate THEP without contaminating granulosa cells. The steroidogenic capacities of "impure" THEP and "pure" theca-epithelium (P-THEP) were then compared. Basal amounts of Pq Were produced when Pg was added to P-THEP, whereas significantly higher amounts were produced in the presence of impure THEP. No significant conversion of P-5 or P-4 to 17 alpha-OHP4 occurred following culture with pure or impure THEP layer. Results suggest that the enzyme activity necessary to metabolize AD --> T is localized in the THEP, whereas the metabolic capacities to convert P-5 --> AD and 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.