STEROIDOGENESIS IN FUNDULUS-HETEROCLITUS .3. EVIDENCE FOR INVOLVEMENT OF CAMP AND PROTEIN-SYNTHESIS IN THE GONADOTROPIC MODULATION OF OVARIAN-STEROID PRODUCTION AND AROMATASE-ACTIVITY

The role of cAMP and the involvement of protein synthesis in ovarian steroidogenesis and aromatase activity of the killifish Fundulus heteroclitus were examined by using isolated prematurational follicles (1.2–1.4 mm diameter) cultured in vitro. Addition of dibutyryl cAMP (dbcAMP) or cAMP in combination with an inhibitor of its catabolism [3‐isobutyl‐1‐methylxanthine (IBMX)] promoted a dose‐dependent increase of media 17α‐hydroxy,2β‐dihydroprogesterone (17α‐OH,20β‐DHP), testosterone (T), and 17β‐estradiol (E2) levels, comparable to the response invoked by a F. heteroclitus pituitary extract (FPE). In addition to steroid production, there was a two‐fold increment in follicular 3H‐leucine incorporation following FPE treatment, probably due to stimulation of protein synthesis. Cycloheximide blocked both 3H‐leucine incorporation and steroid production in a dose‐related manner. FPE also promoted a dose‐related enhancement (more than an additive effect) of E2 accumulation when T was included in the culture media. Results obtained with dbcAMP or cAMP + IBMX in combination with T were essentially the same as with FPE and T, indicating that FPE or increasing levels of cAMP are able to modify aromatase activity. When the endogenous production of E2 caused by FPE or cAMP (dbcAMP or cAMP + IBMX) treatment was suppressed by the use of trilostane (an inhibitor of Δ5‐3β‐hydroxysteroid dehydrogenase), the conversion of exogenous T to E2 was still significantly enhanced. However, no such stimulatory effect on aromatase activity was observed under the same conditions in the presence of cycloheximide. Taken together these results suggest that FPE‐stimulated follicular steroid production by large, prematurational follicles is likely mediated by an intracellular increase in cAMP levels and involves protein synthesis, including that of aromatase. Also, the production of maturation‐initiating steroid by F. heteroclitus follicles does not appear to be affected by physiological levels of E2, unlike the situation reported for other fish and amphibians. Copyright © 1990 Wiley‐Liss, Inc., A Wiley Company