Changes in Content of Cytochrome P45017α, Cytochrome P450scc, and 3-Hydroxy-3-Methylglutaryl CoA Reductase in Developing Rat Ovarian Fotlicles and Corpora Lutea: Correlation with Theca Cell Steroidogenesis1

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Abstract
The following study was undertaken to determine which hormones (luteinizing hormone (LH) and prolactin, PRL) and enzymes (cytochrome P45017.alpha., nicotinamide dinucleotide phosphate [NADPH]-cytochrome P450 reductase, 3-hydroxy-3-methylglutaryl [HMG] CoA reductase, cholesterol side-chain cleavage cytochrome P450 [P450scc], and adrenodoxin) were associated with the regulation of androgen biosynthesis by developing rat follicles and corpora lutea in vivo as well as by thecal explants maintained in culture. Immunoblots of soluble cell extracts of small antral (SA), preovulatory (PO), and luteinizing (PO + human chorionic gonadotropin [hCG], 7 h) follicles, newly formed corpora lutea (PO + hCG, 24 h), and corpora lutea isolated on Day 15 of pregnancy, demonstrated that cytochrome P45017.alpha. was low in SA follicles, selectively increased 4-fold in PO follicles, and decreased to less than 10% within 7 h after hCG. Filter hybridization assays using a 32P-labeled cytochrome P45017.alpha. cDNA probe demonstrated that changes in the content of P45017.alpha. mRNA exhibited a pattern similar to that of the enzyme. Conversely, immunoblots for other microsomal enzymes either exhibited no change (NADPH cytochrome P450 reductase) or a transient increase after the hCG surge (HMG CoA reductase), whereas the mitochondrial enzymes either increased markedly in association with luteinization (cytochrome P450scc) or were increased in a more tranient manner (adrenodoxin). The LH-induced loss of cytochrome P45017.alpha. in vivo was not associated with loss of androgen biosynthesis when luteinizing theca were placed in culture in medium containing either LH or LH and PRL, suggesting that other hormones, or the presence of other cell types, are required to maintain the decrease in cytochrome P45017.alpha. in vivo. Conversely, the LH-induced increase in cytochrome P450scc in vivo was associated with the maintenance of elevated progesterone production by theca in culture, suggesting that cytochrome P450scc may be constitutively expressed in luteinized theca. Thus, thecal cell cytochrome P45017.alpha. and the regulation of its content and mRNA by LH are pivotal to the biosynthesis of androgens, the obligatory precursors for estradiol biosynthesis and the consequent development of prevoulatory follicles. The molecular basis for the different effects of low versus elevated concentration of LH on cytochrome P45017.alpha., as well as cytochrome P450scc, remain to be determined.