RELATIVE ROLES OF THECA AND GRANULOSA-CELLS IN OVARIAN FOLLICULAR STEROIDOGENESIS IN THE AMPHIBIAN, RANA-NIGROMACULATA

Using frog ovarian follicles in vitro we assessed the role of the thec a/epithelium (THEP) layer and granulosa cells in the production of ste roids. The THEP layer and granulosa cell-enclosed oocytes (GCEOs) were separated from ovarian follicles of the frog, Rana nigromaculata, by microdissection. Intact follicles (IFs), THEP layer, or GCEOs were cul tured for 6 hr in amphibian Ringer's in the presence or absence of Var ious steroid precursors (100 ng/ml) or of frog pituitary homogenate (F PH, 0.05 gland/ml). The amounts of progesterone (P-4), 17 alpha-hydrox yprogesterone (17 alpha-OHP4), androstenedione (AD); testosterone (T), and estradiol (E(2) secreted into the medium by the different types o f follicular components during culture were measured by radioimmunoass ay. Addition of pregnenolone resulted in a marked increase in P-4 conc entrations produced by GCEOs (1858 pg/follicle) and IFs (1549 pg/folli cle), but a much smaller increase in P-4 occurred in the THEP layer (4 27 pg/follicle). Likewise, addition of P-4 stimulated greater producti on of 17 alpha-OHP4 by GCEOs (659 pg/follicle) and by IFs (877 pg/foll icle) than by the THEP layer (330 pg/follicle). Exogenous 17 alpha-OHP 4 markedly increased AD levels secreted by GCEOs (1139 pg/follicle) an d by IFs (841 pg/follicle), but not by the THEP layer (<15 pg/follicle ). In contrast, AD addition resulted in the secretion of markedly elev ated levels of T by the THEP layer (2326 pg/follicle) and IFs (3652 pg /follicle) as compared to the GCEOs (408 pg/follicle). Addition of T m arkedly increased E(2) concentrations produced by GCEOs (378 pg/follic le) and by IFs (673 pg/follicle), but not by the THEP layer (< 12 pg/f ollicle). Without addition of FPH or steroid precursors, none of the f ollicular components examined produced significant amounts of the ster oids measured (<20 pg/follicle). Denuded oocytes incubated in the pres ence of exogenous substrates or FPH secreted nearly nondetectable amou nts of steroids. These data suggest that the main sites for synthesis of P-4, 17 alpha-OHP4, AD, and E(2) are granulosa cells and those of T are the theca/epithelium layer in amphibian ovarian follicles. (C) 19 94 Academic Press, Inc.