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  • Title: Developmental and hormonal regulation of rat theca-cell differentiation factor secretion in ovarian follicles.
    Author: Magarelli PC, Zachow RJ, Magoffin DA.
    Journal: Biol Reprod; 1996 Aug; 55(2):416-20. PubMed ID: 8828848.
    Abstract:
    We have recently presented data demonstrating that preantral follicles secrete a peptide (or family of peptides) that stimulates ovarian theca-interstitial cell (TIC) androgen production by an LH-independent mechanism. The purpose of the study reported here was to study the gonadotropin and developmental regulation of this thecal differentiating factor(s) (TDF) and to determine whether follicle-conditioned medium (FCM) containing TDF bioactivity could stimulate LH receptor and steroidogenic enzyme mRNA expression in TIC. Preantral follicles devoid of theca were obtained by limited enzymatic dispersal of 26-day-old rat ovaries. Follicles were cultured (5 follicles/well) in 96-well plates containing serum-free medium to generate FCM containing bioactive TDF. To bioassay for TDF activity, isolated TIC were cultured (2 days) with 50% FCM; then androsterone production was measured by RIA. Recombinant FSH (rFSH, 0.3-100 mlU/ml) increased TDF bioactivity in a dose-dependent fashion, stimulating maximum androsterone production (20 ng/ml) at 30 mlU/ml. To determine the time course of the production of TDF bioactivity, FCM was collected from follicle cultures treated with and without rFSH at 1, 6, 12, 18, 24, 30, 36, 42, and 48 h. FCM from follicles cultured without rFSH caused a progressive increase in androsterone production to a peak (8 ng/ml) at 18 h followed by a decline to baseline by 48 h. A similar time course was observed for the first 18 h with the rFSH-treated FCM, but androsterone production continued to increase to a level twice that of the untreated FCM (18 ng/ml) at 36 h of culture. In the presence of 100 mlU/ml of rFSH, TDF bioactivity was produced by preantral follicles with > or = 2 layers of granulosa cells but not by small antral follicles, preovulatory follicles, or corpora lutea, demonstrating that production of TDF bioactivity is developmentally regulated. To determine whether FCM could stimulate mRNA expression in TIC, LH receptor, cholesterol side-chain cleavage (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), and 17 alpha-hydroxylase (P450(17) alpha) mRNAs were measured by reverse transcription polymerase chain reaction assays. FCM stimulated LH-receptor, P450scc, 3 beta-HSD, and P450(17) alpha mRNAs above controls. Our data demonstrate that the production of TDF bioactivity is increased by FSH during a specific stage in follicular development when the theca interna is rapidly differentiating, but its production stops when the follicle develops an antrum. Treatment of TIC with FCM stimulates the expression of the mRNAs coding for LH receptors and the steroidogenic enzymes P450scc, 3 beta-HSD, and P450(17) alpha, mimicking the events that occur during normal thecal differentiation. Thus, it seems likely that TDF is involved in the regulation of initial thecal differentiation in preantral follicles.
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