Regulation of cholesterol side-chain cleavage enzyme activity by gonadotropin in rat corpus luteum

The locus of gonadotropin-induced acute stimulation of pregnenolone production by cholesterol side-chain cleavage (CSCC) enzyme containing cytochrome P450 (cytP450scc) was examined in rat corpus luteum. Mitochondria were isolated from pseudopregnant rat ovaries after treatment with different doses of human CG (hCG) (25-200 IU) for 30 min; Electron Paramagnetic Resonance (EPR) spectra of high spin cholesterol complex of cyt P450scc (type I high spin EPR signal) and the cyt P450scc activity were determined. hCG treatment increased the formation of type I EPR spectra compared to that obtained with saline-treated controls, and pretreatment with cycloheximide (30 mg/kg BW) before hCG abolished this increase. The magnitude of type I EPR signal diminished with increasing pH over the range of 6.2-7.3. The type I EPR signal increased with doses of hCG and correlated well with the pregnenolone production. Aminoglutethimide treatment (competitive inhibitor of CSCC) before hCG injection led to an increased accumulation of cholesterol in inner mitochondrial membranes with a corresponding decrease in the outer membrane cholesterol, and cycloheximide treatment inhibited this accumulation. This suggests that the transport of cholesterol to inner mitochondrial membranes from outer membranes is regulated by hCG. In addition, gonadotropin also regulates the redistribution of cholesterol within the inner mitochondrial membranes.     

Biosynthesis of cholesterol side-chain cleavage cytochrome P-450 in human fetal adrenal cells in culture

An immunoglobulin G fraction from antiserum raised against cholesterol side-chain cleavage cytochrome P-450 (cytochrome P-450sec) purified from bovine adrenocortical mitochondria cross-reacted with human fetal adrenal cytochrome P-450sec in an Ouchterlony double diffusion system. This property of the immunoglobulin G fraction was used for the immunoisolation of cytochrome P-450sec of human fetal adrenal cells in culture. The molecular weight of the immunoisolate from [35S] methionine-labeled human fetal adrenal cells was similar to that of purified bovine adrenocortical cytochrome P-450sec (49,000). However the molecular weight of cytochrome P-450sec immuno-isolated from an in vitro translation system programmed by human fetal adrenal RNA was approximately 54,000, about 5,000 larger than that of the immunoisolate from radiolabeled cells. The rate of synthesis of cytochrome P-450sec by human fetal adrenal cells in culture, when incubated with ACTH (10(-6) M) for 36 h, was 2.5-fold greater than that by fetal adrenal cells incubated in the absence of ACTH. This result suggests that, as in the case of bovine adrenal cells, cytochrome P-450sec of human fetal adrenal tissue is synthesized as a larger molecular weight precursor which presumably undergoes processing to the mature form during translocation into the mitochondria. This synthesis is stimulated by ACTH, which is the putative trophic factor regulating human fetal adrenal steroidogenesis.     

The mitochondrial environment is required for activity of the cholesterol side-chain cleavage enzyme, cytochrome P450scc.

Steroidogenesis is initiated by the conversion of cholesterol to pregnenolone by mitochondrial cytochrome P450scc [cholesterol, reduced-adrenal-ferredoxin:oxygen oxidoreductase (side-chain-cleaving); EC 1.14.15.6]. Several subsequent steroidal conversions occur in the endoplasmic reticulum (ER), but the last step in the production of glucocorticoids and mineralocorticoids again occurs in the mitochondria. Although cellular compartmentalization of steroidogenic enzymes appears to be a feature of all steroidogenic pathways, some reports indicate that cholesterol can be converted to pregnenolone outside the mitochondria. To investigate whether P450scc can function outside the mitochondria, we constructed vectors producing P450scc and various fusion enzymes of P450scc with electron-transport proteins and directed their expression to either the ER or the mitochondria. Whether targeted to mitochondria or to the ER, plasmid vectors encoding P450scc and fusion proteins of P450scc with either mitochondrial or microsomal electron-transport proteins produced immunodetectable protein. When expressed in mitochondria, all of these constructions converted 22-hydroxycholesterol to pregnenolone, but when expressed in the ER none of them produced pregnenolone. These results show that P450scc can function only in the mitochondria. Furthermore, it appears to be the mitochondrial environment that is required, rather than the specific mitochondrial electron-transport intermediates.     

Vasoactive intestinal peptide induces the synthesis of the cholesterol side-chain cleavage enzyme complex in cultured rat ovarian granulosa cells.

Vasoactive intestinal peptide (VIP) has been identified in ovarian nerves and stimulates steroid secretion from immature ovaries. To gain insight into its mechanism of action, the effect of VIP on the synthesis of the cholesterol side-chain cleavage enzyme complex was studied in ovarian granulosa cells from immature estrogen-primed rats. The cells were cultured for 48 hr in serum-free medium; the proteins were labeled with [35S]methionine; and the synthesis of cytochrome P-450, iron-sulfur protein, and NADPH:iron-sulfur protein reductase was evaluated by electrophoretic analysis after immunoisolation with polyclonal antibodies directed against the bovine adrenal enzymes. VIP at concentrations ranging from 0.001 to 1 microM stimulated 3- to 5-fold the synthesis of cytochrome P-450 and iron-sulfur protein. Peptide NH2-terminal histidine, COOH-terminal isoleucine, which has greater than 50% sequence homology of VIP, stimulated the synthesis of both proteins at approximately 50% of VIP effectiveness. Secretin, another member of the glucagon-secretin family of peptides, which has only 30% sequence homology to VIP, was without effect. Similar results were observed with the NADPH:iron-sulfur protein reductase. VIP-induced synthesis of the cholesterol side-chain cleavage enzyme complex was accompanied by a dose-related increase in cAMP accumulation and progestin formation. It is concluded that VIP regulates the synthesis of the ovarian cholesterol side-chain cleavage enzyme complex, which catalyzes the rate-limiting reaction in progesterone biosynthesis, and that the VIP effect is at least partially mediated through cAMP. It is suggested that a stimulatory action of VIP on the synthesis of ovarian progesterone may contribute to regulating the functional development of the ovary.     

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.     

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.     

The influence of age on steroidogenic enzyme activities of the rat adrenal gland: enhanced expression of cholesterol side-chain cleavage activity

The ability of isolated adrenocortical cells to secrete corticosterone in response to ACTH challenge declines as rats age, but the site or mechanism(s) of this impairment is still unknown. To test the functionality of steroidogenic capacity per se, we measured the key enzyme activities involved in corticosterone biosynthesis. We also measured the mitochondrial cytochrome P-450 content and nonsteroidogenic enzymes specific for subcellular fractions. Mitochondria and microsomal fractions were isolated from the adrenals of 2-, 12-, and 18-month-old animals and used for various enzyme measurements. Mitochondrial side-chain cleavage enzyme activity (nanomoles per min mg protein-1) increased from a mean of 0.43 +/- 0.06 in 2-month-old rats to 1.26 +/- 0.11 and 1.51 +/- 0.06 in 12- and 18-month old rats, respectively. After incubation with 5-cholesten-3 beta,25-diol (25-hydroxycholesterol; 25 micrograms/ml) side-chain cleave activity rose to 5.0 +/- 0.6, 12.4 +/- 1.2, and 16 +/- 1.4 nmol min-1 mg protein-1 in adrenal mitochondrial fractions from 2-, 12-, and 18-month-old rats, respectively. In contrast, mitochondrial cytochrome P-450 content did not vary with advancing age. Microsomal delta 5-3 beta-hydroxysteroid dehydrogenase-delta 5-delta 4-isomerase activities were similar in 2- and 12-month-old rats, but 21-hydroxylase (nanomoles per min mg protein-1) activity was significantly increased in 12-month-old rats (2-month-old, 5.2 +/- 0.2; 12-month-old, 7.7 +/- 0.5). Finally, mitochondrial 11 beta-hydroxylase was comparable in both age groups. In addition, activities of mitochondrial nonsteroidogenic enzymes, such as monoamine oxidase, amytal insensitive NADH cytochrome c reductase, cytochrome c oxidase, succinate dehydrogenase, and malate dehydrogenase, did not change with age. It appears from the evidence presented that the activities of the steroidogenic enzymes are not responsible for the diminished capacity in corticosterone production seen with aging in the rat.     

Inhibition of the activities of P450 cholesterol side-chain cleavage and 3 beta-hydroxysteroid dehydrogenase and the amount of P450 cholesterol side-chain cleavage by testosterone and estradiol-17 beta in hen granulosa cells

We have found that androgens and estradiol-17 beta (E2) produced by theca cells suppress progesterone (P4) secretion by granulosa cells of the domestic hen in a dose-dependent manner. Furthermore, testosterone (T) and E2 inhibited the conversion of cholesterol to pregnenolone (P5) and of P5 to P4, respectively. The aim of this study was to determine if T and E2 suppress P4 biosynthesis by changing activities of the cytochrome P450 cholesterol side chain cleavage (P450scc) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) (Exp I) and the amount of P450scc (Exp II). Granulosa layers of the largest follicle of two to four hens were obtained 22 h before ovulation, pooled, and isolated granulosa cells were prepared. In Exp I, the specific activities of the P450scc and 3 beta-HSD were measured in mitochondrial and microsomal proteins of granulosa cells, respectively, in the presence of T or E2 (0-10 microM). Addition of T to mitochondrial proteins increased the Michaelis-Menten constant (Km) with no change in the maximum velocity (Vmax) of the P450scc, which suggests competitive inhibition (Ki = 30.9 microM), whereas E2 had no effect on Km and Vmax of the P450scc. Likewise, addition of E2 to microsomal proteins increased the Km with no change in the Vmax of the 3 beta-HSD, which suggests competitive inhibition (Ki = 15.1 microM), whereas T had no effect on Km and Vmax of the 3 beta-HSD. In Exp II, granulosa cells (3 x 10(5)/3 ml.tube) were incubated for 0-12 h in triplicate for each combined treatments of 25-OH-cholesterol (8 microM) and cyanoketone (10 microM), T, or E2 (0-10 microM) in the presence or absence of LH (25 ng). Protein content and P5 secretion were measured and the amount of P450scc was determined by Western blot analysis. Incubation of granulosa cells with T decreased the amount of the P450scc in granulosa cells cultured for 12 h and P5 secretion in granulosa cells cultured for 3 h or longer (P less than 0.05), without a change in protein content and cell viability. Our results suggest that P4 production by granulosa cells is suppressed by T and E2 acting as competitive inhibitors of the P450scc and 3 beta-HSD, respectively, and by T decreasing the amount of the P450scc. We conclude that steroidogenesis in the follicle of the chicken is regulated through the interaction of theca and granulosa layers.     

Modulation of aromatase and P450 cholesterol side-chain cleavage enzyme activities of human placental cytotrophoblasts by insulin and insulin-like growth factor I

The placenta is the primary source of estrogens and progesterone during pregnancy. Because pregnant diabetic women are reported to have lower serum estrogen and higher progesterone concentrations than nondiabetic pregnant women, we studied the roles of insulin and insulin-like growth factor I (IGF-I) in the regulation of human cytotrophoblastic aromatase and P450 side-chain cleavage enzyme (P450 SCC) activities. Incubation of cytotrophoblasts with insulin or IGF-I for 24 h significantly inhibited the conversion of androstenedione to estrogens by approximately 20-40%. Insulin and IGF-I suppressed aromatization at doses as low as 20 and 10 ng/ml, respectively. Insulin's suppressive effect was demonstrable only after 18-22 h of incubation, suggesting an effect of insulin on aromatase protein mass rather than on aromatase activity. Cytotrophoblasts pretreated with insulin for 24 h possessed 23-30% less aromatase activity than control cells, as quantitated directly by the specific release of 3H2O from [3H]androstenedione, indicating that insulin inhibited estrogen synthesis rather than increased estrogen catabolism. Insulin's suppressive effect on aromatase was not due to a toxic effect of insulin, since incubates exposed to insulin for 24 h showed no decrease in cell number, cellular DNA content, or cellular protein content compared to control incubates. Also, insulin's suppression of aromatization was not due to increased cAMP phosphodiesterase activity, since cotreatment with 1 mM (Bu)2cAMP did not alter insulin's suppressive effect. Blockade of the IGF-I receptor of cytotrophoblasts with alpha IR-3, a monoclonal anti-IGF-I receptor antibody, prevented the suppression of aromatase activity by IGF-I, but did not alter insulin's inhibitory effect. This suggests that the two hormones inhibit aromatization via activation of their specific receptors and not by cross-association. Insulin treatment did not affect P450 SCC activity, whereas IGF-I treatment significantly stimulated P450 SCC activity by 19-36%, as measured by the conversion of 25-hydroxycholesterol to progesterone. These studies indicate that insulin exerts a selective inhibitory effect on cytotrophoblastic aromatase activity, whereas IGF-I inhibits aromatase activity but stimulates P450 SCC activity. Since pregnant diabetic women manifest peripheral hyperinsulinemia, and IGF-I levels in fetal cord sera from diabetic pregnancies are elevated, these observations may help explain the lower serum estrogen and elevated progesterone levels associated with diabetic pregnancy.     

Regulation of mRNAs encoding the steroidogenic acute regulatory protein and cholesterol side-chain cleavage enzyme in the elasmobranch interrenal gland

The rate-limiting and regulated step in steroidogenesis, the conversion of cholesterol to pregnenolone, is facilitated by the steroidogenic acute regulatory protein (StAR) and cytochrome P450 cholesterol side-chain cleavage (P450scc). We have isolated cDNAs encoding StAR and P450scc from the Atlantic stingray, Dasyatis sabina, and characterized the steroidogenic activity of the encoded proteins using a heterologous expression system. Green monkey kidney (COS-1) cells cotransfected with D. sabina StAR and human P450scc/adrenodoxin reductase/adrenodoxin fusion (F2) constructs produced significantly more pregnenolone than cells transfected with the F2 construct alone. COS-1 cells transfected with a modified F2 construct (F2DS) in which human P450scc is replaced by D. sabina P450scc had higher rates than cells transfected with D. sabina P450scc alone. In other vertebrates, the stress peptide adrenocorticotropic hormone (ACTH) elicits its effects on corticosteroidogenesis in part through regulation of StAR and P450scc mRNAs. In vitro incubation of D. sabina interrenal tissue with porcine ACTH significantly increased intracellular cAMP and corticosteroid production. As demonstrated by quantitative PCR, ACTH also induced significant increases in mRNA abundance of both StAR and P450scc. Our results suggest that, as in higher vertebrates, chronic ACTH-induced glucocorticoid synthesis in elasmobranchs is mediated by regulation of primary steroidogenic mRNAs. This study is the first to demonstrate steroidogenic activity of an elasmobranch P450scc protein and express a composite elasmobranch steroidogenic pathway in a heterologous cell line. Also, the regulation of StAR and P450scc mRNAs has not previously been demonstrated in elasmobranch fishes.     

Effects of epidermal growth factor on the synthesis of the cholesterol side-chain cleavage enzyme complex in rat ovarian granulosa cells in primary culture

The effect of epidermal growth factor (EGF) on the synthesis of the components of the cholesterol side-chain cleavage enzyme complex (SCC) was studied in rat ovarian granulosa cells. The cells were cultured for 48 h in the presence or absence of EGF (15 ng/ml) and/or FSH (50 ng/ml) after which proteins were radiolabeled by incubation with [35S]methionine followed by immunoprecipitation of newly synthesized P-450scc or adrenodoxin (ISP) with polyclonal antibodies directed against the corresponding proteins from bovine adrenal cortex. In addition the action of EGF on the level of translatable RNA for P-450scc was evaluated using a cell-free translation system programmed with RNA isolated from treated and untreated cells, followed by immunoisolation of newly synthesized proteins. Immunoisolated proteins were separated by polyacrylamide-gel electrophoresis, visualized by fluorography and quantified by densitometry. EGF stimulated progesterone formation by the cells 3-fold and potentiated the FSH-induced stimulation of progesterone formation, but had no effect on cAMP accumulation. EGF also stimulated the synthesis of P-450scc and ISP, and enhanced the FSH-induced synthesis of P-450scc and ISP in a concentration-dependent fashion with a maximal stimulation attained at concentrations ranging from 1.0 to 100 ng/ml. No appreciable changes in the induction pattern were observed when EGF and dibutyryl cyclic AMP (Bt2cAMP) were added together, as compared to when Bt2cAMP was added alone. Neither treatment affected the synthesis of the constitutive mitochondrial enzyme, F1-ATPase. Immunoisolation of P-450scc from the proteins synthesized in a rabbit reticulocyte in vitro translation system programmed with RNA isolated from EGF- and/or FSH-treated cells, revealed that EGF enhanced the FSH-stimulated synthesis of the precursor form of P-450scc.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of the cholesterol side-chain cleavage cytochrome P-450 and adrenodoxin mRNAs in cultured choriocarcinoma cells.

Cytochrome P-450scc (P-450scc) catalyzes the cholesterol side-chain cleavage reaction, a rate-limiting enzymatic step for progesterone synthesis in trophoblastic and other steroidogenic cells. Adrenodoxin is the iron/sulfur protein donating electrons to P-450scc during this reaction. We examined the effects of cholera toxin (CT), an activator of adenylate cyclase, and 12-O-tetradecanoylphorbol acetate (TPA), a phorbol ester protein kinase C activator, on the levels of mRNAs encoding P-450scc and adrenodoxin in JEG-3 choriocarcinoma cells. CT induced in a concentration- and time-dependent manner P-450scc and adrenodoxin mRNA levels to 8-fold and 1.5-fold above that of control, respectively. TPA also increased P-450scc and adrenodoxin mRNA levels about 3-fold and 1.5-fold above that of control, respectively. Epidermal growth factor (EGF) was found to weakly induce P-450scc mRNA accumulation with a maximal 20% stimulation above basal levels. The effects of CT and TPA were apparently additive on both mRNAs. The protein synthesis inhibitor cycloheximide diminished basal, CT-, TPA-, and EGF-stimulated P-450scc mRNA accumulation whereas the opposite was observed for the adrenodoxin mRNA. Insulin-like growth factor I (IGF-I) appeared to have no effect on either mRNA. These data indicate that: (1) the accumulation of P-450scc and adrenodoxin mRNAs is mainly controlled by the cyclic adenosine 3',5'-monophosphate (cAMP)-dependent pathway but their stimulation by TPA- and EGF-induced signals may also play a weaker synergistic role; (2) the protein synthesis inhibitor cycloheximide inhibits basal, CT-, TPA- and EGF-stimulated P-450scc mRNA levels while it increases the expression of adrenodoxin mRNA suggesting that in the malignant trophoblasts these two enzyme mRNAs are differentially controlled.     

Luteinizing hormone induction of the cholesterol side-chain cleavage enzyme in cultured immature rat Leydig cells: no role of insulin-like growth factor-I?

Long-term inductive effects of luteinizing hormone (LH) on cholesterol side-chain cleavage (CSCC) enzyme activity were studied, using cultured Leydig cells isolated from 21-day-old rats. Particular reference was given to the role of insulin-like growth factor-I (IGF-I) as an autocrine or paracrine modulator or as an essential extracellular mediator of LH action. The CSCC enzyme activity was measured using an excess of 22(R)-hydroxycholesterol as substrate to saturate the enzyme, and inhibitors of pregnenolone metabolism to concentrate all the products of the enzyme reaction in pregnenolone. The rate of sterol conversion into pregnenolone (CSCC enzyme activity) reflected the amount of cytochrome P-450scc (P-450scc), as was shown by Western blotting. In cells cultured without LH, the CSCC enzyme activity decreased to 10% on day 7 of the culture period. In the presence of various doses of LH ranging from 0.01 to 100 ng/ml, the CSCC enzyme activity also diminished during the first 3 days of culture, but during the following days, the amount of CSCC enzyme was stimulated by LH. In contrast to the absence of any LH effect on the activity of the CSCC enzyme during the first days of the culture, the endogenous steroid production (no added 22(R)-hydroxycholesterol) could be stimulated at least 10-fold by high doses of LH. When LH (1 ng/ml) was added to cells which had been cultured for 7 days without hormones, CSCC enzyme activity was elevated 8-fold after 4 days of exposure of LH. These effects of LH could be mimicked by dbcAMP (0.5 mM). No evidence could be provided that IGF-I plays any role in the LH induction of the CSCC enzyme; neither the addition of exogenous IGF-I or analogs that do not bind to IGF-I binding proteins (IGFBPs) nor the inactivation of endogenous IGF-I action (through binding to IGFBP and antibodies to IGF-I or via masking of IGF-I receptor by antibodies) could influence the LH induced CSCC enzyme activity. The present data raise the question under which conditions IGF-I is capable of modulating Leydig cell steroidogenesis.     

Evaluation of inhibitors for 17beta-hydroxysteroid dehydrogenase type 1 in vivo in immunodeficient mice inoculated with MCF-7 cells stably expressing the recombinant human enzyme

17Beta-hydroxysteroid dehydrogenase (17HSD1) is an enzyme activating estrone (E1) to estradiol (E2). In the present study, a mechanistic animal model was set up for evaluating putative inhibitors for the human enzyme in vivo. Estrogen-dependent MCF-7 human breast carcinoma cells were stably transfected with a plasmid expressing human 17HSD1. These cells formed estrogen-dependent tumors in immunodeficient mice. In the optimized model, tumor sizes were decreased in both ovariectomized and intact vehicle-treated mice, whereas they were maintained or slightly increased in mice supplemented 2 weeks with an appropriate dose of the 17HSD1-substrate E1. Tumor sizes in mice treated with 0.1 micromol/kg/d of E1 were reduced by administering 5 micromol/kg/d of different 17HSD1-inhibitors and a 86% reduction in size was detected with the most potent inhibitor. A dose-response relationship in the inhibitory effect of this compound further confirmed the validity of the model for testing the drug candidates in vivo.     

Proliferating human granulosa-lutein cells in long term monolayer culture: expression of aromatase, cholesterol side-chain cleavage, and 3 beta-hydroxysteroid dehydrogenase

The development of long term culture conditions with which to study the regulation of expression of aromatase, cholesterol side-chain cleavage enzyme, and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in human granulosa-lutein cells is described in this report. Conditions have been established for the dispersal, growth, freezing, and storage of functional human granulosa cells isolated from preovulatory follicles of women undergoing laparoscopy for gamete intrafallopian tube transfer and in vitro fertilization procedures. Optimal growth conditions for human granulosa-lutein cells were determined by plating cells at a low density and testing the capacity of a variety of culture conditions to support growth. A combination of fetal bovine serum (FBS), horse serum, and the serum substitute UltroSer G was found to increase cell number to maximal levels, 8- to 10-fold higher than with sera alone. Human granulosa-lutein cells grown under these conditions had a doubling rate of 36-40 h and were morphologically distinct from human theca interna cells grown under similar conditions. Human granulosa-lutein cells treated with forskolin retracted and rounded up, whereas cultures of human ovarian theca interna cells or human fibroblasts treated similarly did not retract. Human granulosa-lutein cells were grown for successive passages and transferred to serum-free medium containing forskolin, LH, hCG, or cholera toxin. Addition of these agents resulted in a time- and dose-dependent increase in aromatase activity and progesterone secretion. In these studies FSH treatment was found not to increase aromatase activity. In a study of the time course of 3 beta HSD activity in the absence of forskolin under serum-free conditions, it was found that 3 beta HSD activity increased 3-fold during the 72-h treatment period. Forskolin-stimulated 3 beta HSD activity also increased in a time-dependent manner, with levels in treated cells 3-fold higher than those in control cells. Northern analysis performed on total RNA obtained from forskolin- or hCG-stimulated granulosa-lutein cells confirmed that the increase in aromatase activity was associated with a corresponding increase in levels of mRNA specific for aromatase cytochrome P-450. Levels of mRNA encoding cholesterol side-chain cleavage cytochrome P-450 were similarly increased in cells treated with forskolin compared with unstimulated values at each of the time points investigated. Under serum-free conditions in the absence of stimulation, the 3.4-kilobase band of aromatase cytochrome P-450 mRNA was detectable.(ABSTRACT TRUNCATED AT 400 WORDS)