Hormone stimulated steroid biosynthesis in granulosa cells studied with a fluorogenic probe for cytochrome P-450 scc

Abstract

The regulation of steroidogenesis by luteinizing hormone (LH) was studied in granulosa cells during follicular development using a fluorescent reporter assay based on the metabolism of a fluorescent probe specific for cytochrome P-450 scc (cholesterol side-chain cleavage enzyme). Intact granulosa cells or mitochondria were obtained from the first (F 1) second (F 2) and third (F 3) largest preovulatory follicles of the hen ovary and incubated with the fluorogenic substrate. Metabolism of this substrate by cytochrome P-450 scc generates the highly fluorescent resorufin anion (the fluorescent reporter). In both mitochondria and intact granulosa cells, incubated with the fluorescent substrate, an increase in resorufin fluorescence was observed and the increase was greater in samples derived from F 1 than in samples from F 2 or F 3. In cells, LH added simultaneously with the P-450 scc substrate significantly increased resorufin fluorescence above control values in a time- and dose-dependent manner up to 2–3 h after the incubation was initiated. Forskolin and 8-bromo-cAMP also stimulated metabolism of the P-450 scc substrate significantly by 15 min. When granulosa cells were preincubated with LH before exposure to the P-450 scc substrate resorufin fluorescence was significantly attenuated compared to controls (not exposed to LH in the preincubation period). The decrease in resorufin fluorescence observed when cells were pretreated with LH, may be due to the release of cholesterol from endogenous pools and its competition with the exogenous fluorescence for the substrate P-450 scc enzyme. In granulosa cells that were preloaded with the P-450 scc substrate, the stimulatory effect of LH treatment remained constant from 30 min to 2 h after hormone addition. The results show that this fluorescent probe can be used in a rapid assay for the continuous measurement of the acute effects of hormone agonists on cholesterol conversion to pregnenolone in steroidogenic cells.