Side-chain Cleavage Cytochrome P450scc Research Articles

Multiple Steroidogenic Cell Populations in the Thecal Layer of Preovulatory Follicles of the Chicken Ovary*

The thecal layer of the preovulatory follicle of the chicken ovary produces primarily androgens and estrogens. However, the precise cellular sites of androgen and estrogen synthesis in the thecal layer have not been identified. Therefore, our aims were 1) to identify steroidogenic cells in the thecal layer of the preovulatory follicles by localizing specific steroidogenic enzymes in these cells by immunocytochemistry, and 2) to confirm that these cells which contained the specific steroidogenic enzymes secreted the expected steroid in a short term cell incubation. Follicles were collected 2 h after oviposition and prepared for immunocytochemistry and short term cell incubation. Immunocytochemistry for cholesterol side-chain cleavage cytochrome P450 (P450SCC), 17 alpha-hydroxylase cytochrome P450 (P450C17), and aromatase cytochrome P450 (P450AROM) was performed to localize pregnenolone-, androgen-, and estrogen-producing cells, respectively, on frozen sections and paraffin sections of the five largest preovulatory follicles. Interstitial cells showed immunoreactivity for both P450SCC and P450C17, whereas a specific cell population of the theca externa, hereafter termed aromatase cells, showed immunoreactivity for P450AROM. Furthermore, fibroblasts in the theca externa indicated immunoreactivity for P450C17. The immunoreactivity of P450C17 and P450AROM enzymes in specific cells in the theca externa appeared to decrease with follicular maturation. The third largest, fourth largest, and fifth largest follicles were selected for short term cell incubations because the immunoreactivity for P450 enzymes in the thecal layer of these follicles was high. Isolated theca interna cells, theca externa cells, and a combination of interna and externa cells were incubated with/without ovine LH (oLH) for 3 h. The medium was assayed for progesterone, testosterone, and 17 beta-estradiol by RIAs. Incubation of theca interna cells with oLH increased progesterone and testosterone production in a dose-dependent manner. However, we did not observe any production of progesterone and testosterone by theca externa cells. Theca externa cells produced 17 beta-estradiol, and its production was increased significantly when theca interna and externa cells were coincubated in the present of oLH. Based on these data, we propose a multiple cell theory for steroidogenesis in the thecal layer of preovulatory follicles of the chicken ovary which states that interstitial cells in the theca interna produce progestins and androgens, fibroblasts in the theca externa may function as an additional site for the conversion of progestins to androgens, and aromatase cells in the theca externa require androgens as substrate to produce estrogens.

Multiple Mechanisms for Regulation of 3β-Hydroxysteroid Dehydrogenase/Δ5→Δ4-Isomerase, 17α-Hydroxylase/C17–20Lyase Cytochrome P450, and Cholesterol Side-Chain Cleavage Cytochrome P450 Messenger Ribonucleic Acid Levels in Primary Cultures of Mouse Leydig Cells*

The regulation of mRNA levels for delta 5-3 beta-hydroxysteroid dehydrogenase/delta 5----delta 4-isomerase (3 beta HSD), 17 alpha-hydroxylase/C17-20 lyase cytochrome P450 (P450(17 alpha] and cholesterol side-chain cleavage cytochrome P450 (P450scc) was studied in primary cultures of mouse Leydig cells. Treatment of Leydig cells with 8-bromo-cAMP (cAMP) was essential for expression of P450(17 alpha) mRNA, but not for 3 beta HSD. Treatment with cAMP caused a decrease in basal levels of 3 beta HSD mRNA. The addition of aminoglutethimide (AG), an inhibitor of cholesterol metabolism, to the cAMP-treated cultures resulted in increased expression of both 3 beta HSD and P450(17 alpha) mRNA levels. The addition of testosterone or the androgen agonist mibolerone to cAMP- plus AG-treated cultures reduced 3 beta HSD and P450(17 alpha) mRNA to levels comparable to those observed when cells were treated with cAMP only. The glucocorticoid dexamethasone reduced both basal and cAMP- plus AG-induced increases in 3 beta HSD mRNA, but not in P450(17 alpha) mRNA. Estradiol at a concentration of 1 microM had no effect on cAMP- plus AG-induced 3 beta HSD or P450(17 alpha) mRNA levels. The role of protein synthesis in mediating the cAMP induction of 3 beta HSD, P450(17 alpha), and P450scc was investigated. The addition of cycloheximide (10 micrograms/ml) to cAMP-treated cultures for 24 h completely suppressed both constitutive and cAMP-induced 3 beta HSD mRNA levels. Cycloheximide also repressed cAMP-induced levels of P450(17 alpha) to 12% of levels observed in the absence of cycloheximide. In sharp contrast, 24-h treatment with cycloheximide did not suppress cAMP induction of P450scc mRNA, but reduced basal levels by approximately 50%. A time course of induction by cAMP (50 microM) of P450(17 alpha) and P450scc mRNA showed very similar rates of increase in P450(17 alpha) and P450scc mRNA, with the greatest increase occurring between 12 and 24 h of treatment. The results of the study demonstrate that in normal mouse Leydig cells steady state levels of mRNA for 3 beta HSD, P450(17 alpha), and P450scc are differentially regulated. cAMP is required for maximal levels of all three mRNAs. There is high constitutive expression of 3 beta HSD and P450scc mRNA, while expression of P450(17 alpha) mRNA is absolutely dependent on cAMP stimulation. Endogenously produced testosterone negatively regulates the expression of cAMP-induced P450(17 alpha) and 3 beta HSD, while the glucocorticoid dexamethasone negatively regulates 3 beta HSD and P450scc.(ABSTRACT TRUNCATED AT 400 WORDS)

Interleukin-1 Inhibits Cholesterol Side-Chain Cleavage Cytochrome P450 Expression in Primary Cultures of Leydig Cells*

Interleukin-1 (IL-1) is a potent inhibitor of Leydig cell function. We have reported that IL-1 inhibited hCG-induced cAMP and testosterone formation. In the present study we evaluated the effect of IL-1 on Leydig cell cholesterol side-chain cleavage cytochrome P450 (P450scc) mRNA levels. P450scc is the rate-limiting enzyme for Leydig cell steroidogenesis. Highly purified Leydig cells were prepared from adult Sprague-Dawley male rats (55-65 day-old) using the combination of elutriation and Percoll gradient. Purified Leydig cells were then cultured with or without IL-1 beta (1-100 ng/ml) and recombinant human monocyte-derived IL-1 receptor antagonist (250 ng/ml) for 24 h. hCG (10 ng/ml), 8-bromo-cAMP (0.1 mM), or 4 beta-phorbol 12 beta-myristate 13 alpha-acetate was then added, and cultures were continued for an additional 6 h. P450scc mRNA levels of Leydig cells were very low to undetectable after 24 h in culture and could be stimulated by the addition of either hCG (10 ng/ml) or 8-bromo-cAMP (0.1 mM), but the addition of 4 beta-phorbol 12 beta-myristate 13 alpha-acetate had no effect. P450scc mRNA levels increased as early as 2 h after the addition of hCG. Furthermore, cycloheximide (1 microgram/ml) markedly blocked hCG-induced P450scc mRNA expression. This indicates that synthesis of a labile new protein(s) is required for the induction of P450scc mRNA by hCG. IL-1 beta inhibited hCG-stimulated testosterone formation and P450scc mRNA expression in a dose-dependent manner. The inhibitory effects of IL-1 beta could be reversed by the concomitant addition of IL-1 receptor antagonist. Our results suggest that P450scc mRNA levels of Leydig cells are modulated by IL-1. This may be one mechanism that could explain the inhibitory effects of IL-1 on Leydig cell steroidogenesis.

Effect of Activin A on Dehydroepiandrosterone and Testosterone Secretion by Primary Immature Porcine Leydig Cells1

In the present study, we evaluated the effect of the homodimer activin A on immature porcine Leydig cell functions in primary culture. Activin A (0.5-100 ng/ml) reduced hCG-stimulated dehydroepiandrosterone (DHEA) accumulation in a dose- and time-dependent manner, with a maximal inhibitory effect (58% decrease) at 20 ng/ml (8 x 10(-10) M). Activin A was found not to control steroidogenesis, either through a modulation of the gonadotropin LH/hCG binding or low-density lipoprotein cholesterol binding and internalization. However, activin A significantly decreased pregnenolone (p less than 0.002) and DHEA (p less than 0.001) formation (evaluated in the presence of 10(-5) M of WIN 24540, an inhibitor of 3 beta-hydroxysteroid dehydrogenase/isomerase [3 beta-HSDI]activity) in Leydig cells maximally stimulated with hCG (3 ng/ml, 3 h) or incubated in the presence of 22R-hydroxycholesterol (5 micrograms/ml, 2 h). These findings indicate that activin A probably exerts a partial inhibitory effect on cholesterol side-chain cleavage cytochrome P450 (P450scc) activity. On the other hand, activin A significantly (p less than 0.001) enhanced the conversion of exogenous pregnenolone and DHEA (500 ng/ml) but not of progesterone and androstenedione (500 ng/ml) into testosterone, suggesting that activin A potentially enhances 3 beta-HSDI activity in Leydig cells. Activin A action on 3 beta-HSDI activity was found to be closely related to that of transforming growth factor-beta 1 (TGF beta 1), since both activin A (20 ng/ml) and TGF beta 1 (2 ng/ml) induced a comparable and non-additive increase in 3 beta-HSDI activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of CYP11A (P450SCC) and CYP17 (P450(17) alpha) gene expression in bovine luteal cells in primary culture

Cholesterol side chain cleavage cytochrome P450 (P450SCC) and 17 alpha-hydroxylase cytochrome P450 (P450(17) alpha) are key enzymes involved in steroid hormone biosynthesis. The 5'-flanking regions of the corresponding bovine genes have recently been characterized in the adrenal-derived Y1 cell line and specific DNA sequences, required for basal and cAMP-dependent gene expression, were identified. In order to investigate the molecular mechanisms controlling P450SCC and P450(17) alpha gene expression throughout the ovarian cycle, we devised an electroporation protocol to transfect bovine luteal cells in primary culture with specific chimeric DNA constructs. Transfection of such cells with reporter gene constructs containing 186 base pairs (bp) or more of the CYP11A (P450SCC) 5'-regulatory region resulted in cAMP-responsive reporter gene expression. Reporter gene constructs containing 101 bp or less of this regulatory region were expressed neither in the presence nor in the absence of forskolin. Thus, expression of the CYP11A gene in bovine luteal cells in primary culture appears to be controlled by a cis-acting element located between -186 to -101 bp. Transfection of bovine luteal cells in primary culture with reporter gene constructs containing increasing deletions of the bovine CYP17 (P450(17) alpha) 5'-regulatory region clearly demonstrated that none of the sequences were capable of promoting significant reporter gene expression, neither in the presence nor absence of forskolin. Since no 17 alpha-hydroxylase activity and no specific mRNA encoding P450(17) alpha have been detected in the bovine corpus luteum, the failure of expression of the P450(17) alpha reporter gene constructs mimics that of the endogenous gene. These results demonstrate that the regulation of CYP11A gene expression in bovine luteal cells is carried out by the same cis-acting element (-183/-101) which serves this role in the adrenal Y1 cell line. On the other hand, expression of the CYP17 gene in bovine luteal cells appears to be completely repressed, due to the absence of specific positive transcription factor(s) or the presence of negative regulatory factor(s). Thus, transfection experiments using bovine luteal cells in primary culture represent a first step towards the elucidation of molecular mechanisms underlying the regulation of cytochrome P450 gene expression throughout the ovarian cycle.

Expression of mRNA species encoding steroidogenic enzymes in the rat ovary

We have examined the levels of expression of mRNA species encoding cholesterol side-chain cleavage cytochrome P-450 (P-450scc), 17 alpha-hydroxylase cytochrome P-450 (P-450(17 alpha), aromatase cytochrome P-450 (P-450AROM) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in rat ovaries throughout the oestrous cycle, during pregnancy and in immature animals treated with pregnant mare serum gonadotrophin (PMSG). Total or poly(A)(+)-enriched RNA was prepared from adult rat ovaries throughout the oestrous cycle, from immature rat ovaries 24 and 48 h after treatment and from adult rat ovaries on days 10, 14, 17 and 21 of gestation. Expression of the mRNA species was examined by Northern analysis using specific [32P]cDNA probes. During the oestrous cycle P-450scc mRNA of approximately 1.9 kb was detected at low levels, while 3 beta-HSD mRNA of 1.7 kb was in relatively high abundance throughout the oestrous cycle. While P-450(17) alpha mRNA of 1.9 kb and P-450AROM of 2.7, 2.2 and 1.7 kb were highly abundant during dioestrus, pro-oestrus and oestrus, the levels of these mRNA species decreased markedly to be nearly undetectable during metoestrus. During pregnancy there was considerably more variation in the expression of the mRNA species examined. Expression of P-450scc mRNA was at low, but detectable, levels until day 14, thereafter expression increased to high levels (day 14-21 of gestation). Levels of P-450(17) alpha mRNA on day 10 of gestation were lower than at pro-oestrus during the oestrous cycle and decreased further on days 14 and 17. Expression of 3 beta-HSD was decreased on day 10, but on days 14, 17 and 21 of gestation high mRNA levels were detectable. Ovarian expression of the three P-450AROM species was dramatically increased between days 14 and 17 of pregnancy, but declined by day 21. In immature rats, P-450scc mRNA was detected at low levels in unstimulated animals and increased markedly after treatment with PMSG, while subsequent treatment with human chorionic gonadotrophin (hCG) had a minimal effect on expression. Expression of P-450(17) alpha mRNA was high in unstimulated immature and PMSG-treated rats, but diminished after treatment with hCG. All three P-450AROM mRNA species were undetectable in ovaries from unstimulated immature animals; however, induction of all three was observed in PMSG-treated rats, but this expression decreased to undetectable levels upon subsequent administration of hCG.(ABSTRACT TRUNCATED AT 400 WORDS)

Rat cholesterol side-chain cleavage cytochrome P-450 (P-450scc) gene. Structure and regulation by cAMP in vitro.

The entire rat P-450scc gene has been cloned, positions/sequences of the exon-intron boundaries (I-IX) are described and 940 base pairs (bp) of 5'-flanking DNA have been sequenced, compared to mouse, bovine and human genes, and analyzed by functional assays. Primer extension analysis mapped the transcription start site 32 bp upstream of the initiator methionine codon. The rat P-450scc promoter was ligated to the human growth hormone (GH) gene yielding p-940RsccGH. This rat P-450scc fusion gene and a mouse gene (p-1500MsccGH) were transiently transfected into primary cultures of rat granulosa cells and Y1 adrenal cells. In the Y1 cells primer extension analysis showed that the rat P-450sccGH gene was expressed at lower basal levels than that of the mouse gene but showed greater stimulation (4-8-fold) in response to 8-bromo-cyclic AMP than the mouse (3-4-fold). Similar results were obtained when the fusion genes were transfected into primary cultures of rat granulosa cells and production of GH was measured in the media of the cells stimulated with forskolin. Furthermore, we document that gonadotropins (follicle-stimulating hormone/luteinizing hormone) can induce luteinization of granulosa cells in vitro, that this process is associated with constitutive maintenance of P-450scc mRNA in the absence of hormones/cAMP, that these events associated with luteinization are differentiation-stage specific and occur only in granulosa cells of preovulatory follicles but not in small antral follicles, that the process can be inhibited by cycloheximide if the protein synthesis inhibitor is present during the first 6 h of exposure to luteinizing hormone but not if added for short durations (3-5 h) later, and that once luteinization is induced P-450scc mRNA expression and progesterone biosynthesis are not strictly dependent on cAMP. Thus, the P-450scc gene is regulated by both cAMP-dependent and cAMP-independent mechanisms, each of which are associated with a specific stage of granulosa cell differentiation. The DNA domains involved in regulating these two diverse processes remain to be determined. Although there was remarkable sequence homology among rat, mouse, bovine, and human genes within 70 bp of the transcriptional start site, no other sequence similarities revealed conserved functional domains among the four genes. Although some cAMP-responsive element-like sequences are present in the rat gene, these were not conserved in the other species; including the mouse which showed high sequence homology with the rat throughout 900 bp of 5'-flanking DNA. Thus, the cAMP domains specific to this and other steroidogenic genes remain to be clearly identified.

The Biosynthesis of Cholesterol Side-Chain Cleavage Cytochrome P-450 in the Rabbit Corpus Luteum Depends Upon Estrogen*

To gain a better understanding of the luteotropic action of estrogen, we have investigated the effect of estrogen on the synthesis of the enzyme, cholesterol side-chain cleavage cytochrome P-450 (P-450scc) in the rabbit corpus luteum. Using an established protocol, rabbits were treated with estradiol, and the estradiol was then withdrawn on day 9 of pseudopregnancy, which caused an 88% fall in serum progesterone within 48 h. In other rabbits, estradiol was replaced at 48 h which stimulated a 6.6-fold increase in serum progesterone concentration within the next 24 h. Luteal tissues were incubated with [35S]methionine and homogenized, and a mitochondrial fraction lysate was obtained. Equal trichloroacetic acid-precipitable radioactivity was taken for immunoprecipitation using a well-characterized polyclonal antiserum against bovine adrenal P-450scc. The immunoisolated proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and radioactivity was visualized by autofluorography. The results indicate that the rate of synthesis of P-450scc in 48 h-estradiol withdrawn animals was markedly reduced, and by 72 h of withdrawal was barely detectable. When estradiol was reintroduced, the synthesis of P-450scc was increased. Despite the prominent changes in P-450scc synthesis, immunoblotting revealed only a minimal (approximately 30%) decrease in relative P-450scc content by 72 h after estradiol withdrawal. Analyses of DNA and protein contents of luteal tissues revealed an increase in DNA per mg luteal tissue, a decline in total tissue protein/DNA ratio, but no change in mitochondrial fraction protein/DNA ratio after estrogen withdrawal. The results indicate that de novo synthesis of P-450scc in the corpus luteum is sensitive to estrogen; however, the estrogen-sensitive rate-limiting step(s) for steroidogenesis are at other sites in the steroid biosynthetic pathway.

Glucocorticoid and Cyclic Adenosine 3′5′-Monophosphate-Mediated Induction of Cholesterol Side-Chain Cleavage Cytochrome P450 (P450SCc) in MA-10 Tumor Leydig Cells. Increases in mRNA Are Cycloheximide Sensitive*

The regulation of cholesterol side-chain cleavage enzyme (P450scc) was investigated in MA-10 tumor Leydig cells. We recently demonstrated that the constitutive and cAMP-stimulated expression of P450scc in normal mouse Leydig cells is negatively regulated by glucocorticoids. We now report that glucocorticoids have the opposite effect in MA-10 cells causing a 1.7-fold increase in the rate of P450scc synthesis and a 2.1-fold increase in the amount of P450scc mRNA. Treatment of MA-10 cells with 10 microM 8-bromo-cAMP (8-Br-cAMP) (cAMP) resulted in a 1.7-fold increase in P450scc synthesis and a 3-fold increase in P450scc mRNA. Combined treatment with dexamethasone and cAMP resulted in additive increases in synthesis (2.8-fold) and mRNA (5.3-fold). Increases in de novo synthesis and mRNA levels were reflected by modest increases in the amount of immunoreactive P450scc enzyme protein. Dexamethasone-mediated stimulation in synthesis and accumulation of P450scc mRNA were blocked by the antiglucocorticoid RU-486. Cycloheximide blocked both cAMP- and dexamethasone-induced increases but had no effect on constitutive levels of P450scc mRNA. Treatment of MA-10 cells with 10 microM 8-Br-cAMP had no effect on cell morphology and stimulated progesterone accumulation to a minor degree. Treatment of MA-10 cells with 1 mM 8-Br-cAMP resulted in cell rounding and loss of cells from culture dishes. The results of this study demonstrate that: 1) dexamethasone increases P450scc de novo synthesis and mRNA levels in MA-10 tumor Leydig cells, opposite to the effect in normal Leydig cells; 2) dexamethasone- and cAMP-stimulated increases occur via distinct mechanisms; 3) and synthesis of protein factor(s) is required to mediate the action of both dexamethasone and cAMP.

Alteration in the expression of genes for cholesterol side-chain cleavage enzyme and 21-hydroxylase by hypophysectomy and ACTH administration in the rat adrenal

The changes in steady-state levels of mRNA for cholesterol side-chain cleavage cytochrome P-450 (P-450scc) and steroid 21-hydroxylase cytochrome P-450 (P-450c21) caused by hypophysectomy and ACTH treatment were determined in rat adrenals. Hypophysectomy caused marked decreases in adrenal weight and total RNA per gland. Administration of ACTH resulted in increases in adrenal weight and total RNA. A significant correlation between the amount of RNA and adrenal weight was observed. Both P-450scc and P-450c21 mRNAs were decreased by hypophysectomy and increased by ACTH treatment. P-450scc mRNA decreased to 20% and P-450c21 mRNA to 76% of control values 1 day after hypophysectomy. ACTH caused a significant increase in P-450scc mRNA after 3 h. However, a significant increase in P-450c21 mRNA was observed 12 h after administration of ACTH. These results are concordant with previous studies in vitro utilizing cultured adrenocortical cells. Moreover, the induction of steady-state levels of P-450scc mRNA was faster than that observed by other investigators in studies in vitro. These results may indicate that integrity of the adrenal gland in vivo is important for the action of ACTH.

Effect of LH injections on testicular steroidogenesis, cholesterol side-chain cleavage P450 mRNA content and Leydig cell morphology in hypogonadal mice

Hypogonadal (hpg) mice have a congenital deficiency of hypothalamic gonadotrophin-releasing hormone (GnRH) and the gonads consequently lack exposure to gonadotrophins during development. We injected male hpg mice with LH for 10 days to investigate whether LH alone can stimulate normal steroidogenesis in these animals. Control animals had an inactive interstitium and very few germ cells. Testicular content of androgens was undetectable by radioimmunoassay in control animals unless a single injection of LH was given 1 h before death, when androgens were just detectable. Control testes incubated in vitro with [3H]pregnenolone demonstrated that without gonadotrophin stimulation pregnenolone was metabolized only to progesterone in significant amounts. Assay for cholesterol side-chain cleavage cytochrome P450 (P450scc) mRNA showed basal expression in saline-treated hpg mouse testis. LH treatment induced hypertrophy and hyperplasia of Leydig cells and division of germ cells. Testicular androgen content increased significantly, with testosterone and androstenedione as the major androgens. LH-treated testes incubated with [3H]pregnenolone in vitro had a greater synthetic capacity for testosterone, suggesting an increase in 17 alpha-hydroxylase/C17-20-lyase activity. Basal and human chorionic gonadotrophin-stimulated androgen production in vitro increased markedly following LH treatment to levels previously described in the normal adult animal. LH treatment caused a rapid and transient increase in the hybridization of P450scc mRNA which was sevenfold greater than that of saline-treated controls when the animals were killed 1 h after the last injection but fell to control levels within 24 h of cessation of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II inhibits luteinizing hormone-stimulated cholesterol side chain cleavage expression and stimulates basic fibroblast growth factor expression in bovine luteal cells in primary culture.

Angiotensin II has been identified immunohistochemically in the ovaries of both rats and humans. Here we present evidence that angiotensin II (an extremely vasoactive agent in a wide range of tissues) may be involved in the regulation of the major steroidogenic enzyme in the ovary, cholesterol side chain cleavage cytochrome P-450 (P-450scc), as well as of basic fibroblast growth factor (bFGF), which has been implicated as an angiogenic factor in the bovine corpus luteum. We have used primary cultures of bovine luteal cells to examine the effect of angiotensin II and its receptor antagonist, saralasin, on expression of mRNA encoding bFGF as well as on progesterone production and the expression of mRNA encoding cholesterol side chain cleavage cytochrome P-450 (P-450scc). Neither angiotensin II nor saralasin when added alone to the culture medium had any effect on basal progesterone production. Luteinizing hormone (LH) caused a 15-fold increase in progesterone accumulation after 24 h of exposure which was reduced to 5-fold in the presence of angiotensin II. This appeared to be receptor-mediated in that although saralasin alone had no effect on LH-stimulated progesterone accumulation, it significantly reversed the inhibition by angiotensin II. This pattern was mirrored by the levels of mRNA encoding P-450scc, i.c., LH induced the highest levels of expression of this message, these levels were reduced by angiotensin II, and saralasin partially overcame this reduction. Levels of mRNA encoding bFGF were elevated by both LH and angiotensin II. Treatment with saralasin, however, resulted in complete inhibition of bFGF mRNA expression in the presence of both LH and angiotensin II. These results suggest a role for angiotensin II to mediate the action of LH as a regulator of bFGF expression and hence, potentially, angiogenesis. Local production of angiotensin II might also contribute to the refractoriness of luteal progesterone secretion to LH at the time of luteal regression.

Regulation of Aromatase mRNA and Estradiol Biosynthesis in Rat Ovarian Granulosa and Luteal Cells by Prolactin

Regulation by PRL of aromatase (P450arom) mRNA and protein and estradiol (E) biosynthesis was examined in granulosa cells during early stages of luteinization in vitro and in vivo. PRL caused a dose-dependent (10-1000 ng/ml) decrease in P450arom mRNA and E biosynthesis (greater than 99%) in luteinized rat granulosa cells in vitro, even when the cells were cultured in the presence of insulin and hydrocortisone (hormones known to synergize with PRL to induce proteins in mammary tissue) or in the presence of forskolin (a nonhormonal stimulator of cAMP). PRL also prevented the marked increases in aromatase mRNA and E biosynthesis stimulated by FSH and forskolin in nonluteinized preovulatory granulosa cells in culture. These effects of PRL on granulosa cells in culture were specific for aromatase and were not observed for other proteins, such as cholesterol side-chain cleavage cytochrome P450 (P450scc) and alpha 2-macroglobulin. PRL also decreased P450arom mRNA and protein during the early stages of luteinization in vivo. PRL administered to rats beginning day 1 postovulation to mimic hormone release during pseudopregnancy reduced the progressive increase in P450arom mRNA occurring in corpora lutea on days 3-4 in ovulated rats not treated with PRL. CB 154, a dopamine agonist that inhibits pituitary release of PRL, caused P450arom mRNA and protein to decrease 50% if given to pregnant rats on days 8-10 of gestation, but increased P450arom mRNA and protein if given to pregnant rats on days 10-12 of gestation. These diverse effects of PRL in pregnancy suggest that placental factors may modify the response of luteal cells to PRL during gestation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic AMP-dependent and -independent Regulation of Cholesterol Side Chain Cleavage Cytochrome P-450 (P-450scc) in Rat Ovarian Granulosa Cells and Corpora Lutea.: cDNA and deduced amino acid sequence of rat P-450scc

Abstract We report the isolating and sequencing of three cDNA clones encoding rat P-450scc, the nucleotide and protein sequences of which are highly homologous to those of bovine and human P-450scc, especially in the putative heme and steroid binding domains. We document that different molecular mechanisms regulate P-450scc in granulosa cells of preovulatory (PO) follicles prior to and after luteinization. Luteinizing hormone/human chorionic gonadotropin (LH/hCG) and cAMP are obligatory to induce P-450scc mRNA in PO granulosa cells in vivo and in vitro. Once P-450scc mRNA is induced as a consequence of the LH/hCG surge it is constitutively maintained by luteinized cells in vivo (0-4 days) and in vitro (0-9 days) in the absence of gonadotropins, is susceptible to modulation by prolactin and is no longer regulated by cAMP. Exposure to elevated concentrations of hCG in vivo for 5-7 h was required for PO granulosa cells to undergo a functional transition establishing the stable luteal cell phenotype. Transient exposure of PO + hCG (7 h) follicles in vitro to the RNA synthesis inhibitor actinomycin D (1 microgram/ml) or the protein synthesis inhibitor cycloheximide (10 micrograms/ml), for 1-5 h prior to culturing the granulosa cells failed to disrupt the induction of P-450scc mRNA, progesterone biosynthesis, and appearance of the luteal cell morphology. Inhibitors of protein kinase A (Rp-cAMPS; 1-500 microM and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H8); 1-200 microM) added directly to the luteinized cell cultures also failed to alter P-450scc mRNA in these cells, although the cells contain in vivo amounts of mRNA for RII beta, RI alpha, and C alpha, the primary subunits of protein kinase A found in the rat ovary. These data suggest that expression of the P-450scc gene in rat ovarian follicular cells is regulated in a sequential manner by cAMP-dependent and cAMP-independent mechanisms associated with granulosa cells and luteal cells, respectively.

An Adenosine 3′,5′-Monophosphate-Responsive Deoxyribonucleic Acid Element Confers Forskolin Sensitivity on Gene Expression by Primary Rat Granulosa Cells*

The following studies were conducted with the goal of understanding some of the molecular mechanisms by which cAMP alters granulosa cell function. In this regard, we characterized the expression of messages for the cholesterol side-chain cleavage cytochrome P450 (P450scc) enzyme and a type II cAMP-dependent protein kinase subunit (RII beta) in granulosa cells isolated from small antral follicles and cultured in 1% fetal bovine serum-containing medium. Forskolin (FSK) stimulated cAMP production followed by accumulation of RII beta mRNAs, induction of P450scc mRNA, and, finally, progesterone biosynthesis. The regulation of each mRNA displayed a different sensitivity to actinomycin-D treatment. To determine if the modulation of RII beta or the induction of P450scc could be mediated by the enhancer activity of a cAMP-responsive DNA element (CRE), plasmid DNA containing the CRE and TATA box of the human glycoprotein alpha-subunit (alpha G) gene fused to the chloramphenicol acetyl transferase (CAT) gene was introduced into cultured granulosa or interstitial cells, and the ability of FSK to stimulate the expression of the CAT enzyme was measured. When granulosa cells were isolated from preantral/small antral follicles and maintained for at least 5 days in culture, 5 or 10 microM FSK reversibly stimulated expression of the CAT enzyme within 18 h posttransfection. Under similar conditions, interstitial ovarian cells (prepared from the residual ovarian tissue remaining after granulosa cell isolation) were unable to express the template unless the cells were pretreated for 24 h with FSK before transfection. Thereafter, CAT gene expression by interstitial cells was maintained in a FSK-insensitive manner. To determine if cAMP-dependent transcription of the reporter gene required the same sequences that had been characterized for placenta-derived cells, a truncated plasmid lacking the CRE of the alpha G gene was transfected. Under no condition was expression of the CAT gene observed from a CRE-deficient template. The acute transcriptional activation of a CRE/TATA box-containing gene by cAMP in granulosa cells indicated that transcription-activating proteins interacting with the CRE were either present in these cells or were rapidly synthesized after stimulation. To distinguish between these two possibilities, protein synthesis was transiently inhibited after transfection, before the addition of FSK, by incubating the cells for 2 h with 25 micrograms/ml cycloheximide. Cycloheximide treatment alone did not stimulate transcription from the CRE-containing molecule. Incubation with cycloheximide, followed by treatment with FSK, increased CAT activity 2-fold compared to t

Periovulatory Expression of Cholesterol Side-Chain Cleavage Cytochrome P-450 in Cumulus Cells*

Immunocytochemical staining methods were used to examine the appearance of cholesterol side-chain cleavage cytochrome P-450 (P-450scc) in mitochondria of cumulus cells during follicular development. The cumulus-oocyte complexes were isolated from pregnant mare serum gonadotropin (PMSG)/human CG (hCG)-treated 25 day rats and examined in culture. It is shown that P-450scc is not expressed in the cumulus cell earlier than 2-3 h before ovulation. After ovulation, the expression of P-450scc rapidly increased, so that postovulatory cumuli contained ample amounts of the cytochrome. RIA of progestins secreted by the cumulus-oocyte complexes in culture corroborated the immunocytochemical observations. A single administration of LH or PMSG treatment of hypophysectomized rat did not result in P-450scc accumulation. However, this failure of hormonal responses in vivo was not due to lack of available receptors, since both FSH and LH could induce cAMP accumulation and P-450scc when added to isolated cumuli in culture. Therefore, these findings suggest the presence of a putative intraovarian suppressive factor(s) which disappears before ovulation and thus render(s) the cumulus cells permissive for P-450scc responsiveness. An additional intriguing aspect of P-450scc responsiveness to gonadotropins was revealed in experiments showing that 60% of the cultured cumulus complexes failed to accumulate P-450scc in response to hormones, if collected from 21 day animals. Interestingly, those P-450scc negative cumuli were always associated with an oocyte which did not resume meiotic maturation. We may therefore suggest that meiotic incompetence of the oocyte is also accompanied by functional incompetence of its embracing cumulus cells which cannot, for yet unclear reasons, acquire their steroidogenic capacity.

Cell-specific variations and hormonal regulation of immunoreactive cytochrome P-450scc in the rat ovary.

Specific rabbit antibodies to the bovine cholesterol side-chain cleavage cytochrome P-450 (P-450scc) were used to cross-react with the enzyme in the rat ovary. The luteal cells of cyclic, pregnant, and pseudopregnant rats were immunostained. P-450scc was also expressed in the interstitial cells of prepubertal and cyclic adult rats, and in the thecal cells lining the preovulatory follicles. In cyclic females, RU 486 and oestradiol increased the intensity of P-450scc immunostaining. The granulosa cells of ovarian follicles whatever their stage of development, including preovulatory follicles, were not labelled, except after ovulation. The intensity of immunostaining of thecal and interstitial cells decreased during early pregnancy or pseudopregnancy, and disappeared after Day 9, whereas these cells were intensely labelled 24 h after parturition. The immunostaining of thecal and interstitial cells was again detected in 18-day pregnant rats, treated with the antiprogesterone RU 486. It is therefore concluded that both oestradiol and progesterone are involved in P-450scc regulation.

Simultaneous transfection of COS-1 cells with mitochondrial and microsomal steroid hydroxylases: incorporation of a steroidogenic pathway into nonsteroidogenic cells.

Transfected, nonsteroidogenic COS-1 cells derived from monkey kidney are found to be capable of supporting the initial and rate-limiting step common to all steroidogenic pathways, the side-chain cleavage of cholesterol to produce pregnenolone. Endogenous COS-1 kidney cell renodoxin reductase and renodoxin are able to sustain low levels of this activity catalyzed by bovine cholesterol side-chain cleavage cytochrome P450 (P450scc) whose synthesis is directed by a transfected plasmid containing P450scc cDNA. Double transfection with both P450scc and adrenodoxin plasmids leads to greater pregnenolone production and indicates that adrenodoxin plays a role as a substrate for this reaction or that bovine adrenodoxin serves as a better electron donor than the endogenous iron-sulfur protein renodoxin. Also it is found that both the bovine adrenodoxin and P450scc precursor proteins are proteolytically processed upon their uptake by COS-1 cell mitochondria to forms having the same electrophoretic mobility as mature bovine adrenodoxin and P450scc. Following triple transfection of COS-1 cells with P450scc, adrenodoxin, and 17 alpha-hydroxylase cytochrome P450 plasmids, pregnenolone produced in mitochondria by the side-chain cleavage reaction can be further metabolized in the endoplasmic reticulum to 17 alpha-hydroxypregnenolone and dehydroepiandrosterone. Although this functional steroidogenic pathway can be incorporated into this nonsteroidogenic cell type, it is found to be nonresponsive to cAMP, a potent activator of steroid hormone biosynthesis in adrenal cortex, testis, and ovary. Thus the cellular mechanisms necessary to support both microsomal and mitochondrial steroid hydroxylase activities appear not to be tissue specific, whereas the acute cAMP-dependent regulation of steroidogenesis is not present in transformed kidney (COS-1) cells.

Tetradecanoyl phorbol acetate suppresses follicle-stimulating hormone-induced synthesis of the cholesterol side-chain cleavage enzyme complex in rat ovarian granulosa cells.

The effect of tetradecanoylphorbol acetate (TPA) on follicle-stimulating hormone (FSH)-induced synthesis of the cholesterol side-chain cleavage (SCC) enzyme complex was studied in rat ovarian granulosa cells cultured for 48 h in serum-free medium. Cell proteins were radiolabeled with [35S]methionine, followed by immunoprecipitation of cholesterol side-chain cleavage cytochrome P-450 (P-450SCC) as well as the iron-sulfur protein adrenodoxin. Polyacrylamide gel electrophoresis and fluorography of the immunoprecipitates showed that TPA, when added in combination with FSH (50 ng/ml) or dibutyryl cAMP (Bt2cAMP; 1 mM), suppressed the stimulatory effects of these compounds on the synthesis of the SCC components in a concentration-dependent fashion. The effect of TPA was accompanied by decreased progesterone formation and decreased cAMP accumulation. The structural analog of TPA, phorbol-4 alpha-didecanoate, which does not activate protein kinase C (Ca2+/phospholipid-dependent enzyme), had no effect on the FSH- or Bt2cAMP-stimulated synthesis of SCC and progesterone or on cAMP formation. In addition to inhibiting the synthesis of these proteins, TPA greatly reduced the FSH- and Bt2cAMP-induced increase in levels of mRNA encoding the precursor form of P-450SCC. It is concluded that the effect of the phorbol ester TPA to inhibit FSH-stimulated progesterone formation in cultured ovarian granulosa cells of the rat involves decreased synthesis of the components of the SCC enzyme complex due to reduced levels of mRNA encoding the precursor forms of these proteins. The results are indicative that TPA not only inhibits FSH-mediated stimulation of cAMP formation but also may block cAMP-mediated induction of SCC synthesis. It is postulated that the effects of TPA may reflect the physiological role of protein kinase C in the regulation of ovarian steroidogenesis.

Vasoactive Intestinal Peptide Regulates Cholesterol Side-Chain Cleavage Cytochrome P-450 (P-450scc) Gene Expression in Granulosa Cells from Immature Rat Ovaries

Vasoactive intestinal peptide (VIP), a neuropeptide present in ovarian nerves, has been previously shown to induce synthesis of the side-chain cleavage cytochrome P-450 enzyme which catalyzes the conversion of cholesterol to pregnenolone (the rate-limiting step in progesterone synthesis). In the present study we demonstrate, by means of a bovine 3'-specific P-450scc cDNA probe, that this VIP effect is exerted at least partially at the level of gene expression in cultured granulosa cells that were isolated from estrogen-primed, immature rats. The size and level of the 2.0 kilobase P-450scc mRNA species was assessed by Northern blot analysis, while the translatability of this mRNA was assayed by immunoisolation of the 35S-labeled P-450scc precursor protein translated from total RNA of control and stimulated granulosa cells. FSH was much more effective than VIP at increasing P-450scc mRNA concentrations in cultured granulosa cells, whereas secretin treatment was ineffective. The results suggest that, like FSH, the stimulatory effect of the neuropeptide VIP on ovarian progesterone secretion involves regulation of P-450scc gene expression during functional maturation of the prepubertal ovary.