Parameters affecting the elongation block by RNA polymerase II at the SV40 attenuator 1 in vitro.

We have previously suggested that folding of the SV40 attenuator RNA 1 into two hairpin elements leads to a block of transcription elongation. Using site-directed mutagenesis, we created three templates that either strengthened or weakened the proposed hairpin structures. The mutated and wild-type templates were cloned downstream from the adenovirus 2 MLP, and transcription patterns were compared among the templates, in cell-free extracts. In this report, we show that at the standard temperature (30 degrees C) the elongation block occurs at elevated KCl concentrations (0.5-1.2 M KCl) while at high temperature (65 degrees C), although the transcription elongation rate increases, the block to elongation occurs at lower KCl concentrations (less than 0.5 M KCl) as well. Since under the conditions tested the extent of the elongation block is directly dependent on the stability of the hairpin structure of the RNA, as assessed by a comparison of transcription of the wild-type and mutated templates, we suggest that elevated KCl concentrations and high temperatures are favored conditions for optimizing the processes whereby the hairpin structures are recognized by the polymerase as an attenuation signal. Moreover, the present experiments indicate that cellular elongation factors are not directly involved in modulating the extent of the elongation block at the SV40 attenuator 1 in vitro. The SV40 attenuator 1 is the only eukaryotic system known to date which has been shown to have structural characteristics so similar to rho-independent terminator in prokaryotes. We discuss the similarities between the mechanisms which lead to elongation blocks at these eukaryotic and prokaryotic sites.

Cholesterol side-chain cleavage P450 messenger ribonucleic acid: evidence for hormonal regulation in rat ovarian follicles and constitutive expression in corpora lutea.

Using an affinity-purified antibody against cholesterol side-chain cleavage P450 (P450scc) and a human P450scc cDNA probe, 11 rat P450scc cDNA clones were identified and isolated from our rat granulosa cell lambda gt11 cDNA expression library. Two clones were plaque purified and subcloned into pBR322. One of these P450scc cDNA clones, approximately 1.2 kilobases (kb) in size, was used as a probe for Northern and filter hybridization assays to analyze the tissue distribution and hormonal regulation of P450scc mRNA in rat ovarian follicles and corpora lutea. Northern transfers revealed a single P450scc mRNA species about 2.0 kb in size. Filter hybridization assays showed that P450scc mRNA was low in granulosa cells and thecal cells of small antral follicles, was increased in both tissues of preovulatory follicles, and was rapidly (within 7 h) and maximally increased (30-fold) during hCG-induced luteinization. P450scc enzyme and mRNA were also elevated in corpora lutea isolated from pregnant rats (days 4-22 of gestation) and rats 1 day after parturition (day 23). The elevation of P450scc enzyme and mRNA was maintained despite the marked decline in serum progesterone concentrations between days 19-22, suggesting that once P450scc mRNA is induced in luteal tissue it may be constitutively expressed. Administering hormones to granulosa cells in culture and to hypophysectomized immature rats in vivo demonstrated that the induction of P450scc mRNA by FSH in granulosa cells was time, dose, and estradiol dependent. High doses of FSH acting on estradiol-primed cells gave the greatest response. The increase in P450scc mRNA in cultured granulosa cells was also stimulated by forskolin and was directly associated with increased synthesis of cAMP and progesterone accumulation. Thus, whereas the induction of P450scc mRNA in granulosa cells was dependent on hormones and cAMP, the maintenance of P450scc mRNA and P450scc protein in corpora lutea appears to involve constitutive expression of P450scc mRNA.

Immunofluorescent probing of the mitochondrial cholesterol side-chain cleavage cytochrome P-450 expressed in differentiating granulosa cells in culture.

Mitochondria in follicular cells from rat ovaries were visualized in culture by indirect immunofluorescence staining of cholesterol side-chain cleavage cytochrome P-450 (P-450scc). The confinement of the immunofluorescence in the conspicuous mitochondria allowed the design of a very sensitive and quantitative assay to study the modulated expression of the cytochrome in primary cultures of granulosa cells. (1) The induction of P-450scc synthesis was totally dependent upon treatment with FSH. Up to 85% of the cells became immunofluorescently labeled in the presence of FSH, and its induced P-450scc synthesis was inhibited by cycloheximide and alpha-amanitin. The induction of FSH was dose dependent (Kmapp = 35 ng/ml) and time dependent. Prolonged incubation with FSH maintained the high levels of the cytochrome content, despite a desensitized steroidogenic response which developed after 60 h of incubation with FSH. Prolonged FSH treatment also resulted in morphological changes in the induced mitochondria, which became fragmented and globular. (2) Inoculum densities, probably by altering cell shape, substantially affected the extent of P-450scc induction; this was suppressed (80%) at lower culture densities. (3) The immunofluorescent staining also revealed various degrees of cellular competence to express P-450scc. Within a single induced cell, all mitochondria emitted a similar fluorescent signal, but the degree of fluorescence per mitochondrion varied with different cells. The cell-specific information gained by the immunofluorescent technique also allowed the detection of ovarian interstitial cells that slightly contaminate the granulosa cell preparations. Unlike granulosa cells, interstitial cells express and maintain high levels of P-450scc without the need for hormonal induction.

Concerted metabolism of steroid hormones produced by cocultured ovarian cell types.

Ovaries of immature, intact rats were dispersed by collagenase-DNase treatment and cultured in serum-free medium (ovarian cell culture). The hormonal responsiveness of the ovarian cell was compared to that exhibited by pure granulosa cells isolated via needle puncturing. Surprisingly, despite the fact that the majority of the cultured cells should have been comprised of granulosa type, no follicle-stimulating hormone-inducible progesterone or 20 alpha-OH-progesterone (20 alpha-OH-P) could be detected by radioimmunoassay, as typically occurs in cultures of pure granulosa cells. Therefore, in order to unravel the cause for the different responsiveness between the granulosa and the ovarian cell, we applied thin-layer chromatography analysis to follow the metabolic fate of added radioactive pregnenolone to intact monolayers in culture. Such TLC analysis revealed that, after priming with follicle-stimulating hormone, added [3H]pregnenolone was converted to progesterone which was rapidly reduced and finally accumulated as 5 alpha-pregnane-3 alpha,20 alpha-diol(pregnanediol). In addition to pregnanediol, a second class of steroid hormones accumulated in the coculture medium, namely androsterone and 5 alpha-pregnane-3 alpha,17 alpha,20 alpha-triol (pregnanetriol). These latter two were specific products of the ovarian coculture, indicating the presence of theca-interstitial cells bearing 17 alpha-hydroxylase and 17,20-lyase activities. Pregnanediol, rather than progesterone, was found to be the progestin precursor for androgen formation. We thus conclude that due to exchange of steroid metabolites between the cocultured cell types, the final steroid products are different by far from the expected contributions of each individually cultured cell type. Moreover, these findings reveal an additional aspect of the "two-cell theory," suggesting a granulosa-thecal concerted metabolism of progestin steroids, rather than exchange of aromatizable androgens.

Low molecular weight substance from rat ovary induces steroidogenesis in cultured granulosa cells.

Ovaries from immature intact rats contain an apparently low molecular weight substance which mimics the action of follitropin (FSH) on ovarian granulosa cells in culture. Similar to FSH action, the ovarian substance (OS) induced cell-shape changes followed by intensive progestin production. Like FSH action, OS-induced steroidogenesis reversibly ceased upon washing the factor from the cultured cells, and could be blocked in the presence of cycloheximide or alpha-amanitin. Although OS stimulated aromatase activity in granulosa cells, it failed to elicit LH responsiveness in the cultured cells. Androstenedione synergistically augmented OS-induced progestin production and aromatase activity. OS itself synergistically augmented FSH-induced progestin but did not have any effect on FSH-induced aromatase activity. In contrast to FSH action which is mediated via cAMP formation, OS doses which evoked extensive synthesis of progestin products failed to stimulate significant increases in intracellular cAMP accumulation. These results suggest the existence of a putative intraovarian hormone-like substance which can mimic some effects of the gonadotropins on the follicular granulosa cell differentiation and may facilitate FSH action at yet unknown stages of the follicular development.