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The effects of FSH and (Bu)2cAMP on synthesis of the components of the cholesterol side-chain cleavage (SCC) enzyme complex, namely SCC cytochrome P-450 (P-450scc), the iron-sulfur protein adrenodoxin (ISP), and NADPH:ISP reductase (Red), were investigated in granulosa cells obtained from ovaries of immature estrogen-primed rats cultured for up to 72 h in defined medium in the presence or absence of FSH and (Bu)2cAMP. The cells were lysed, and proteins were subjected to polyacrylamide gel electrophoresis, followed by immunoblotting using antibodies specific to bovine adrenocortical P-450scc, ISP, and Red. A time-dependent increase was observed in the specific contents of these three components of SCC, but not of the reference mitochondrial protein, F1-ATPase, upon treatment with FSH or (Bu)2cAMP. The increase in the content of these three enzymes was accompanied by a rise in progesterone and 20 alpha-hydroxyprogesterone production. The synthesis of P-450scc, ISP, and Red increased 3- to 4-fold with time upon FSH or (Bu)2cAMP treatment respectively, as evidenced by pulse labeling of the cell proteins with [35S]methionine, followed by immunoprecipitation. Immunoprecipitation of P-450scc and ISP from an in vitro translation system programmed by RNA isolated from cultured cells revealed that treatment with FSH or (Bu)2cAMP resulted in an increase in the levels of translatable mRNA specific for these proteins, and that the initial products of translation were precursor forms of cytochrome P-450scc and ISP, similar to those observed in bovine adrenal and granulosa cells. It is concluded that in cultured rat ovarian granulosa cells, FSH induces the synthesis of cytochrome P-450scc, ISP, and Red by increasing the content of translatable mRNA coding for the precursor forms of these enzymes and that this action is mediated by cAMP. Furthermore, the effects of FSH and (Bu)2cAMP provide an explanation for the action of these compounds to stimulate progestin synthesis in cultured ovarian cells.