Effects of epidermal growth factor on the synthesis of the cholesterol side-chain cleavage enzyme complex in rat ovarian granulosa cells in primary culture.
The effect of epidermal growth factor (EGF) on the synthesis of the components of the cholesterol side-chain cleavage enzyme complex (SCC) was studied in rat ovarian granulosa cells. The cells were cultured for 48 h in the presence or absence of EGF (15 ng/ml) and/or FSH (50 ng/ml) after which proteins were radiolabeled by incubation with [35S]methionine followed by immunoprecipitation of newly synthesized P-450scc or adrenodoxin (ISP) with polyclonal antibodies directed against the corresponding proteins from bovine adrenal cortex. In addition the action of EGF on the level of translatable RNA for P-450scc was evaluated using a cell-free translation system programmed with RNA isolated from treated and untreated cells, followed by immunoisolation of newly synthesized proteins. Immunoisolated proteins were separated by polyacrylamide-gel electrophoresis, visualized by fluorography and quantified by densitometry. EGF stimulated progesterone formation by the cells 3-fold and potentiated the FSH-induced stimulation of progesterone formation, but had no effect on cAMP accumulation. EGF also stimulated the synthesis of P-450scc and ISP, and enhanced the FSH-induced synthesis of P-450scc and ISP in a concentration-dependent fashion with a maximal stimulation attained at concentrations ranging from 1.0 to 100 ng/ml. No appreciable changes in the induction pattern were observed when EGF and dibutyryl cyclic AMP (Bt2cAMP) were added together, as compared to when Bt2cAMP was added alone. Neither treatment affected the synthesis of the constitutive mitochondrial enzyme, F1-ATPase. Immunoisolation of P-450scc from the proteins synthesized in a rabbit reticulocyte in vitro translation system programmed with RNA isolated from EGF- and/or FSH-treated cells, revealed that EGF enhanced the FSH-stimulated synthesis of the precursor form of P-450scc.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D002478:Cells, Cultured; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000096:biosynthesis* / Q000235:genetics; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D005260:Female; D006107:Granulosa Cells / Q000166:cytology / Q000201:enzymology*; D010088:Oxidoreductases / Q000096:biosynthesis*; D011374:Progesterone / Q000096:biosynthesis; D014176:Protein Biosynthesis / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011919:Rats, Inbred Strains
D012333:RNA, Messenger; D011374:Progesterone; D004815:Epidermal Growth Factor; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D002786:Cholesterol Side-Chain Cleavage Enzyme