Addition Of Aminoglutethimide Research Articles

A Regulatory Mechanism Between Progesterone and Notch Signaling Pathway Maintains the Functionality of the Corpus Luteum from Pregnant Rats.

The Notch signaling pathway includes a family of transmembrane receptors (Notch1-4) that interact with specific ligands (Delta-like family, Jagged1 and Jagged2). This system regulates cell fate decisions, including proliferation, differentiation and apoptosis. The transmembrane receptors are cleaved upon binding of their ligands, leading to the release of the intracellular domain (NICD), which translocates to the nucleus where it functions as a transcriptional coactivator of regulatory genes of cellular fate. This processing requires the activity of two proteases, namely tumour necrosis factor a-converting enzyme and presenilin/gamma-secretase. We have previously shown that Notch1-4 receptors and the ligand Dll4 are expressed in the corpus luteum (CL) of pregnant rats and that the administration of prostaglandin F2 alpha decreases the levels of mRNA and protein expression of these proteins. Furthermore, blocking ovarian Notch system with the gamma-secretase inhibitor DAPT decreases serum levels of progesterone (P4) and increases pro-apoptotic protein expression in the CL. However, the role of P4 in regulating the Notch pathway in the CL of pregnant rats remains unclear. In this study CL′s were isolated from ovaries of rats on day 16 of gestation and cultured for 4 hours in the presence of aminoglutethimide (inhibitor of cytochrome P450scc 0.15 mM), DAPT (gamma secretase inhibitor, 20 uM), both inhibitors and the inhibitors with the addition of P4 (500ng/ml). Luteal function was assessed by measuring P4 levels in the medium by RIA. The CL levels of Notch1-4 and members of the ERK and PI3K/AKT pathways were measured by Western blot. The treatment of CL′s with each inhibitor significantly decreased P4 levels (P < 0.05, n = 5) in the culture medium, effect which was similar in the presence of both compounds. Furthermore, these inhibitors decreased protein levels of Notch1 intracellular fragment (NICD) compared to the control group (P < 0.05, n = 5). However, no significant difference was observed for NICD of Notch4. In addition, ERK and AKT phosphorylation decreased with the addition of aminoglutethimide or/and DAPT in the CL culture medium. These results suggest the existence of a regulatory mechanism between progesterone and the Notch signaling pathway to maintain the functionality of the CL in pregnancy. Supported by: CONICET (PIP1223), Universidad de Buenos Aires (X-218) and ANPCYT (PICT, 2010-0248).

Expression of ghrelin and the ghrelin receptor in different stages of porcine corpus luteum development and the inhibitory effects of ghrelin on progesterone secretion, 3β-hydroxysteroid dehydrogenase (3β-honestly significant difference (HSD)) activity and protein expression

Recent studies have suggested that ghrelin plays a direct role in controlling female reproduction. The aim of the present study was to investigate the mRNA and protein expression of ghrelin and its receptor (via real time PCR, Western blot and immunohistochemistry analysis, respectively) in porcine corpora lutea (CL) collected during early (CL1: 1–2 days after ovulation), middle (CL2: 7–10 after ovulation), and late luteal phase (CL3: 13–15 after ovulation). Ghrelin expression and concentration of both acylated and unacylated forms of ghrelin significantly increased during CL development. Immunohistochemistry analysis shown localization of ghrelin protein in the cytoplasm of large luteal cells. No changes in the expression of the ghrelin receptor were observed. Direct in vitro effects of ghrelin on progesterone (P4) secretion and 3-beta-hydroxysteroid dehydrogenase (3β-honestly significant difference (HSD)) activity, which were measured by the conversion of pregnenolone (P5) to P4, and 3β-HSD protein expression were then analyzed. To assess 3β-HSD activities, mature luteal cells were first cultured for 24 h with ghrelin at 100, 250, 500 and 1000 pg/mL with P5, or with aminoglutethimide (AMG). AMG is an inhibitor of CYP11A1-mediated hydroxylation; an addition of AMG and P5 enabled P4 production to serve as an index of 3β-HSD activity. Inhibitory effects of ghrelin on P4 secretion, 3β-HSD activity and protein expression were observed. In conclusion, the presence of ghrelin and its receptor in porcine corpora lutea and the direct inhibitory effects of ghrelin on luteal P4 secretion and 3β-HSD suggest potential auto/paracrine regulation by ghrelin in the luteal phase of ovary function.

Stimulatory effect of progesterone on the expression of steroidogenic acute regulatory protein in MA-10 Leydig cells.

The steroidogenic acute regulatory protein (StAR), by virtue of its ability to facilitate the intramitochondrial transport of cholesterol, plays an important role in regulating steroid hormone biosynthesis in steroidogenic cells. In agreement with published data, both StAR expression and progesterone production in MA-10 mouse Leydig tumor cells could be stimulated with hCG and 8Br-cAMP. Addition of aminoglutethimide, an inhibitor of cholesterol side chain cleavage (P450(scc)) enzyme, not only resulted in a drastic inhibition of progesterone production but also in an attenuation of StAR expression in response to either hCG or 8-Br-cAMP. Therefore, we addressed the question of whether progesterone, the end product of the steroidogenic cascade in these cells, could be in a position to regulate the StAR gene expression. In MA-10 cells, we report here that progesterone in microgram amounts can induce StAR gene expression in a time- and dose-dependent manner. StAR expression in response to a maximally effective concentration of progesterone of 10 microg/ml was highest at 6 h and started decreasing thereafter. The effect of progesterone on StAR protein and StAR mRNA induction was mimicked by its synthetic analog, progestin R5020, but not by other steroids, including dexamethasone, estradiol, testosterone, and dihydrotestosterone. Dexamethasone, in contrast, was able to inhibit StAR expression in MA-10 cells. Surprisingly, RU486, a potent antagonist of progesterone and glucocorticoid action, had a stimulatory effect on StAR mRNA levels. Reverse transcription-polymerase chain reaction analysis demonstrated the absence of the classical form of progesterone receptor in MA-10 cells. Thus, for the first time, a direct stimulatory effect of a steroid on StAR gene expression has been demonstrated. Furthermore, these results provide a new insight, indicating that progesterone mediates the activation of StAR expression exerted presumably through a novel, nonclassical progesterone receptor in mouse Leydig cells.

Lack of Effect of Aminoglutethimide and Hydrocortisone Added to High‐Dose Bicalutamide for Androgen‐Independent Prostate Cancer

ABSTRACTObjectives: To determine whether hormones derived from the adrenal gland affected the progression of androgen‐independent (AI) prostate cancer for patients already receiving treatment with high‐dose bicalutamide. This was accomplished by using a sequential trial design in which medically or surgically castrated patients with AI prostate cancer whose cancers were progressing while receiving bicalutamide were treated with adrenolytic therapy in the form of aminoglutethimide (AM) and hydrocortisone (HC).Materials and Methods: Sixteen patients with AI prostate cancer were enrolled into this trial. All patients had metastatic disease in the bone, and two patients had additional metastases at other sites. Using prostate specific antigen (PSA) level as an indicator of disease activity, patients initially received bicalutamide therapy, then AM and HC were added to the regimen, and then bicalutamide therapy was discontinued, with each documented PSA level increase (see Figure 1). Clinical Trial Study Design: Patients were treated with sequential hormonal interventions according to the scheme depicted above. imageResults: Four (25%) patients demonstrated PSA level reductions of at least 50% after the initiation of bicalutamide, 150 mg/day. No patient demonstrated a PSA or obvious clinical response to the addition of AM and HC to the regimen. This includes the four patients who were initially sensitive to bicalutamide. Therapy with AM and HC was associated with fatigue and skin rashes. These side effects persisted after the withdrawal of bicalutamide therapy and were therefore attributed to AM and HC therapy.Conclusions: Bicalutamide at high doses seems to effectively block the androgen receptor (AR) from the growth stimulation derived from adrenal hormones. Therefore, the inhibition of adrenal hormone production by AM is not a useful therapeutic maneuver in this setting and is associated with moderate side effects. The activation of the AR pathway in these patients may occur by a mechanism other than stimulation by the androgens derived from the testes or adrenal gland.

Glucocorticoids enhance corticotropin receptor mRNA levels in ovine adrenocortical cells.

We have shown previously that chronic treatment with glucocorticoids enhances both ACTH-induced cAMP production and ACTH- or 8Br-cAMP-induced steroidogenesis of cultured ovine adrenocortical cells. This treatment has been shown to involve an increase in the number of ACTH receptors. The present study aimed to explore the mechanism of this effect of glucocorticoids on ACTH receptors. Ovine adrenocortical cells expressed one major ACTH receptor transcript of 3.6 kb and three minor ones of 4.2, 1.8 and 1.3 kb. Dexamethasone treatment of cultured cells increased the levels of all these transcripts in a time- and dose-dependent manner, with an EC50 of (1.5 +/- 0.6) x 10(-8) M. The mean increase over control with 10(-6) M dexamethasone was 144 +/- 11% (n = 14). This enhancing effect was specific for glucocorticosteroids. The antiglucocorticoid Ru38486 blocked the effect of dexamethasone. Testosterone did not modify, while high concentrations of 17 beta-estradiol decreased, ACTH receptor mRNA levels. Treatment of cells with aminoglutethimide (an inhibitor of steroidogenesis) resulted in a dose-dependent decrease in ACTH receptor mRNA levels, which was prevented by concomitant treatment with dexamethasone. Treatment with ACTH also increased ACTH receptor mRNA levels more than twofold. Addition of aminoglutethimide together with ACTH resulted in a smaller increase than that achieved with ACTH alone. Neither dexamethasone nor ACTH modified ACTH receptor mRNA half-lives. However, these two hormones enhanced the levels of both newly synthesized and total ACTH receptor mRNAs. These results indicate that the positive trophic effect of glucocorticoids on ovine adrenocortical cells involves an enhancement of the transcription rate of the ACTH receptor gene. In addition, they suggest that part of the trophic action of ACTH on ACTH receptors may be mediated by ACTH-induced steroidogenesis.

The role of endozepine in the regulation of steroid synthesis

Endozepine has recently been isolated from various steroid-forming organs. The following article explores the role of endozepine in the regulation of steroid synthesis. Steroid hormone synthesis from cholesterol begins in the inner mitochondrial membrane, where cytochrome P450 converts cholesterol to pregnenolone. Scientists thought that ACTH would stimulate this conversion, but experiments showed no such stimulation. However, addition of aminoglutethimide to block side-chain cleavage caused the expected reaction of ACTH to take place. Next the role of protein synthesis on the actions of ACTH was explored. Then endozepine was isolated from bovine fasciculata based on stimulation of pregnenolone production by freshly prepared mitochondria. After further experimentation it was concluded that endozepine is a peptide with at least two groups of actions: It binds GABAA receptors in the central nervous system, and it increases the mitochondrial synthesis of pregnenolone.

Combined treatment with 4-hydroxyandrostenedione and aminoglutethimide: effects on aromatase inhibition and oestrogen suppression.

The effects of a combination of aminoglutethimide (AG) 1,000 mg daily and 4-hydroxy-androstenedione (4OHA) 500 mg i.m. weekly on peripheral aromatase activity as measured by in vivo radioisotopic tracer methodology and serum oestrogen suppression were investigated in ten post-menopausal women with advanced breast cancer. Patients were treated for a minimum of 4 weeks with 4OHA before addition of AG for a minimum of 6 weeks. Aromatase inhibition was found to be nearly identical in the two treatment situations (92.5 +/- 4.7% and 93.8 +/- 3.8% respectively). There was no further significant suppression of plasma oestradiol or plasma oestrone levels when AG was added to 4OHA treatment (mean decrease of 7.6 +/- 12.1% and 2.8 +/- 12.0% respectively). In contrast, adding AG caused a further suppression of plasma oestrone sulphate (Oe1S) compared with 4OHA monotherapy (mean suppression of 35.2 +/- 9.1%, P < 0.025). This effect on Oe1S may be due to an influence of AG on oestrogen metabolism.

Growth suppression of MCF-7 human breast cancer cells by aromatase inhibitors: A new system for aromatase inhibitor screening

In our previous study we found that MCF-7 cells possess aromatase activity and stimulate estrogen receptor-mediated growth. The pathways through which androgens are converted to estrogens by aromatase and estrogens interact with estrogen receptors contribute significantly to growth stimulation. The administration of aromatase inhibitor results in suppression of growth stimulation by androgens. This system enabled us to assess directly the biological activities of aromatase inhibitors. Aromatase activity was inhibited in a dose-dependent manner by the addition of aminoglutethimide and CGS 16949A, competitive inhibitors, and of 14α-hydroxy-4-androstene-3,6,17-trione and 4-hydroxy-androstenedione, mechanism-based inhibitors. After preincubation with mechanism-based inhibitors, aromatase activity was significantly suppressed, whereas after preincubation with competitive inhibitors, it was adversely increased. These effects were concentration- and time-dependent. Preincubation with competitive inhibitors resulted in augmentation of subsequent androgen stimulation of thymidine incorporation, while preincubation with mechanism-based inhibitors resulted in diminished stimulation by subsequent androgen administration. These results suggest that in MCF-7 cells competitive inhibitors adversely induce aromatase and accelerate the subsequent androgen stimulation of DNA synthesis. Suicide inhibitors are more effective than competitive inhibitors. This system will be useful for aromatase inhibitor screening.

Hormonal regulation of steroidogenic enzyme gene expression in Leydig cells

In normal mouse Leydig cells, steady state levels of mRNA of CYP11A, 3β-hydroxysteroid dehydrogenase Δ 5- > Δ 4-isomerase (3βHSD), and CYP17 are differentially regulated. There is high basal expression of 3βHSD and CYP11A mRNA, while expression of CYP17 mRNA is absolutely dependent on cAMP stimulation. cAMP is required for maximal expression of all three enzymes. The expression of CYP11A in normal mouse Leydig cells is repressed by glucocorticoids. Glucocorticoids also repress both basal and cAMP-induced expression of 3βHSD mRNA, but do not repress the synthesis or mRNA levels of CYP17. cAMP induction of 3βHSD mRNA can be observed only when aminoglutethimide (AG), an inhibitor of cholesterol metabolism, is added to the Leydig cell cultures. The addition of AG also markedly increases cAMP induction of CYP17 mRNA levels. Addition of testosterone or the androgen agonist, mibolerone, to cAMP plus AG treated cultures reduced 3βHSD and CYP17 mRNA levels to levels comparable to those observed when cells were treated with cAMP only. These data indicate that testosterone acting via the androgen receptor represses expression of both CYP17 and 3βHSD. The role of protein synthesis in mediating the cAMP induction of 3βHSD, CYP17 and CYP11A was examined. The addition of cycloheximide, an inhibitor of protein synthesis, to cAMP treated cultures for 24 h completely suppressed both constitutive and cAMP-induced 3βHSD mRNA levels. Cycloheximide also repressed cAMP-induced levels of CYP17 to 12% of levels observed in the absence of cycloheximide. In sharp contrast, treatment for 24 h with cycloheximide did not suppress cAMP induction of CYP11A mRNA, but reduced basal levels by approx. 50%. These data indicate that newly synthesized protein(s) are required for cAMP induction of CYP17 and 3βHSD mRNA levels, but not for CYP11A mRNA. A mouse Cyp17 genomic clone containing the entire coding region plus 10 kb of 5′ flanking region has been isolated. Fragments of 5′ flanking sequences were subcloned into vectors containing the CAT reporter gene and transfected into MA-10 Leydig cells. Transfected cells were treated with cAMP and expression was determined by measuring CAT activity. A cAMP responsive element was identified in a region between -245 and -346 bp relative to the transcription initiation site of Cyp17. Cotransfection into MA-10 Leydig cells of constructs containing 4.5 kb of Cyp17 5′ flanking sequences together with a mouse androgen receptor expression vector demonstrate a dose dependent repression of cAMP-induced Cyp17 transcription by the androgen receptor. Studies with the mouse Cyp11a gene demonstrate that the 5′ flanking region of the gene contains sequences between 2.5 and 5 kb that are necessary for expression of mouse Cyp11a in Leydig cells but not in adrenal cells.

Mitomycin C and Aminoglutethimide in the Treatment of Metastatic Prostatic Cancer: a Phase II Study

In a phase II study, 24 patients with metastatic prostatic cancer were treated with mitomycin C 15 mg/m2 i.v. every 6 weeks, combined with aminoglutethimide 250 mg twice a day. A low dose of 37.5 mg cortisone acetate was supplied daily to compensate for adrenal cortical suppression. A partial response was demonstrated in 4 of 24 evaluable patients with bi-dimensionally measurable metastases. Stable disease occurred in 8 patients over a period of more than 6 months. Within the maximum cumulative dose limit of 2 mg/kg body weight mitomycin C, toxicity was observed in 21 cases, including 2 deaths due to treatment toxicity. The poor response rate and high toxicity suggest that the addition of aminoglutethimide does not enhance the effect of mitomycin C in these patients.

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)

Apolipoprotein E synthesis by cultured human ovarian granulosa cells: regulation by human chorionic gonadotropin and cholesterol

Apolipoprotein E (Apo E) is a surface component of several classes of plasma lipoproteins and functions as a receptor ligand for the low-density lipoprotein (LDL) (B/E) receptor, the hepatic E receptor, and the LDL receptor related protein. In continuing studies of Apo E synthesis by extrahepatic steroidogenic tissue, we have examined Apo E production and its control in cultured human granulosa cells (GC). Human GC obtained at the time of oocyte aspiration for in vitro fertilization were cultured, and the following parameters were measured: progesterone secretion into the medium, Apo E synthesis and secretion, and Apo E messenger ribonucleic acid (mRNA) content of the GC. Human chorionic gonadotropin induces a decrease in Apo E protein synthesis and Apo E mRNA content. The addition of aminoglutethimide (which blocks cholesterol conversion to pregnenolone) and/or LDL (which provides the cell with cholesterol), both result in an increase in Apo E protein synthesis and Apo E mRNA content. These latter findings are in agreement with studies in nonsteroidogenic cells, which show that Apo E production is positively correlated with cell cholesterol content.

Involvement of Protein Kinase C in the Regulation of Cortisol Production by Guinea Pig Adrenocortical Cells

Cytosol of the guinea pig adrenals was found to contain a protein kinase which was dependent on the presence of both calcium and phospholipids (phosphatidylserine and diolein), i.e., calcium/phospholipid-dependent protein kinase (protein kinase C). The peak of protein kinase C was separated from type II cAMP-dependent protein kinase by DE-52 chromatography. 12-0-Tetradecanoylphorbol-13-acetate (TPA) caused dose-dependent increments of cortisol formation without affecting cAMP formation by guinea pig adrenocortical cells as well as angiotensin II did. TPA-activated cortisol production was blocked by the addition of aminoglutethimide and cycloheximide, suggesting that the site of action of TPA might be located at a point before the production of pregnenolone in the mitochondria. Since TPA showed an increase in the cortisol production, protein kinase C may be involved in modulating steroidogenesis in the guinea pig adrenals in addition to the classical cAMP-dependent protein kinase pathway.

The rise in testicular androgens during the first days of treatment with an LHRH agonist in the dog can be blocked by aminoglutethimide or ketoconazole

Up to day 6 of treatment of adult dogs, daily subcutaneous administration of 50 μg of the LHRH agonist [D-Trp 6, des-Gly-NH 2-10]LHRH ethylamide causes up to a 3-fold increase in serum testosterone (T) concentration which is followed by a progressive decrease to castration levels (⩽ 0.2 ng/ml) at later time intervals (up to 21 days, the last time interval studied). Both aminoglutethimide and ketoconazole, two inhibitors of steroid biosynthesis, cause a 30–40% rise in serum T when administered alone. However, either drug administered in combination with the LHRH agonist completely blocks the transient rise in serum T observed when the LHRH agonist is administered alone. On the other hand, the LHRH agonist prevents the secondary rise in steroid secretion observed when either of the two inhibitors of steroid secretion is used alone. Administration of the pure antiandrogen Flutamide alone or in combination with LHRH-A and an inhibitor of steroid biosynthesis does not influence serum T levels. When the serum levels of pregnenolone 17OH-pregnenolone, progesterone, 17-OH-progesterone, dehydroepiandrosterone (DHEA), androstenedione (Δ 4-dione), androst-5-ene-3β,17β-diol (Δ 5-diol), T, dihydrotestosterone (DHT) androstane-3α,17β-diol, androstane-3β.17β-diol and 17β-estradiol (E 2) are analyzed in detail, it can be seen that both aminoglutethimide and ketoconazole not only prevent the rise in serum steroids observed during the first 8 days of treatment with the LHRH agonist but that both compounds enhance the inhibitory effect of the LHRH agonist at later time intervals. A predominant inhibitory effect of ketoconazole is exerted on 17,20-desmolase activity. Aminoglutethimide has little influence on the loss of serum LH bioactivity induced by the LHRH agonist while ketoconazole stimulates the concentration of serum bioactive LH in the absence or presence of simultaneous treatment with the LHRH agonist. The present data clearly demonstrate that aminoglutethimide or ketoconazole can prevent the rise in serum androgens accompanying the first days of treatment with an LHRH agonist in the dog. Moreover, after 3 weeks of treatment, the inhibitory effect of the LHRH agonist on serum androgen levels is enhanced by addition of aminoglutethimide or ketoconazole. Moreover Flutamide does not interfere with the inhibitory action of the LHRH agonist, aminoglutethimide or ketoconazole, thus suggesting that maximal inhibition of androgen action is likely to be achieved by a combination of these drugs

Hydrocortisone alone vs hydrocortisone plus aminogluthethimide: a comparison of the endocrine effects in postmenopausal breast cancer

The endocrine effects of replacement doses of hydrocortisone in postmenopausal women with advanced breast cancer were compared with the same doses of hydrocortisone plus aminoglutethimide. Fifteen patients received aminoglutethimide (AG) 250 mg three times a day plus hydrocortisone (HC) 20 mg twice a day for 2 weeks, then AG was increased to 250 mg four times a day. Another 13 patients received HC alone for 2 weeks, then AG was added. HC alone significantly suppressed oestrone (75% of baseline) and oestradiol (50% of baseline). Addition of AG to these patients produced further oestrone suppression (50% of baseline) significantly greater than HC alone. HC alone suppressed dehydroepiandrosterone sulphate as much as AG + HC. Δ 4-androstenedione ( Δ 4A) and dehydroepiandrosterone (DHA) were suppressed by HC alone. Addition of AG produced a rise of Δ 4A to basal levels. These results show that 3-β-oldehydrogenase is not induced by AG. AG plus HC together from day 1 produced significantly greater oestrone suppression (50% of baseline) than 11C alone. Because high-dose steroids may induce aromatase and replacement doses produced marked peripheral endocrine effects, the use of replacement hydrocortisone should be reassessed in advanced breast cancer.

Progesterone and prostaglandin secretion by ovulated rat cumulus cell-oocyte complexes.

Hormone production (progesterone and prostaglandin) by oocyte-cumulus cell complexes was studied in the postovulatory period. Cumulus-oocyte complexes were collected from oviducts of prepuberal rats after the synchronization of follicle development and ovulation with PMS gonadotropin and hCG injections. Cumulus masses from individual animals were cultured in roller vessels containing Eagles' Minimal Essential Media with or without added heat-inactivated rat sera (5% CO2-air; 37 C). Culture media were collected after 4.5 h of incubation and analyzed by RIA for prostaglandin E2 (PGE), PGF, and progesterone. Extensive progesterone secretion occurred during the culture period and was markedly stimulated by the addition of rat serum; however, cumulus masses did secrete some progesterone in the absence of rat serum and without exogenous gonadotropins. The addition of aminoglutethimide, a steroidogenesis inhibitor, suppressed progesterone secretion but did not alter the secretion of PGF or PGE. Both PGF and PGE were detected in the culture medium, and in all cases, PGE was the predominant PG detected. Indomethacin, an inhibitor of PG synthesis, suppressed PGE and PGF secretion but had no effect on progesterone synthesis. Small amounts of PGs were also detected in nonincubated cumulus-oocyte masses. Hormone secretion in culture was measured after cumulus mass dissociation by enzymatic digestion. Somatic cumulus cells, rather than germ cells, were the primary cellular source of progesterone and PGs. The results demonstrate that cumulus-oocyte complexes can synthesize and secrete steroid and PG hormones subsequent to ovulation, and thus, these masses should be considered functional endocrine tissues. The physiological significance of the endocrine activities of the cumulus-oocyte complex are discussed in terms of oviductal and gametic processes. (Endocrinology 108: 457, 1981)

Effect of follicular steroids on the maturation and fertilization of mammalian oocytes

Pronuclear development was used to measure the effects on ovine oocytes of altering follicular steroidogenesis during maturation in vitro. Follicular steroid secretion was altered using enzyme inhibitors and exogenous steroid supplementation. Abnormalities induced during maturation were measured 24 h after tranfer of oocytes to the oviducts of inseminated hosts. The presence throughout maturation of aminoglutethimide, an inhibitor of the conversion of cholesterol to pregnenolone, reduced steroid secretion to 7% of that in controls and decreased from 77% to 33% the number of normal oocytes. Abnormalities were substantially reduced by the addition of aminoglutethimide during the final 8 h of maturation only. The inhibition of 17 alpha-hydroxylase enzymes with SU10603 reduced oestrogen and testosterone secretion to about 10% of control levels but had no effect on progestin secretion. Only 13% of oocytes matured in the continual presence of SU10603 underwent normal fertilization. The number of oocytes undergoing normal fertilization was increased to about 50% by (i) delaying the addition of SU10603 until the last 8 h of the maturation period or (ii) adding exogenous steroids to follicles cultured with inhibitor from explantation. It is concluded that oocytes require a specific intra-follicular steroid environment for the completion of the full maturation process. Alterations to the steroid profile during maturation induce changes in the oocyte which are expressed as gross abnormalities at fertilization.

Gonadotrophin Stimulation of Progesterone Synthesis by Midpregnancy Mouse Ovarian Cellsin Vitro

The ability of cultured midpregnancy mouse ovarian cells to synthesize progesterone de novo and from oxogenous pregnenolone has been assessed. The conversion of pregnenolone to progesterone is almost completely blocked by cyanoketone, a known inhibitor of delta5,3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity, but is unaffected by aminoglutethimide, which inhibits the cholesterol side-chain cleavage enzyme complex (desmolase). Since there is little metabolism of the formed progesterone, the ability of ovarian cells to convert exogenous pregnenolone to progesterone in vitro reflects the activity of 3beta-HSD in these cells. Cultured ovarian cells are also capable of endogenous progesterone production in the absence of added pregnenolone, although the absolute amount of progesterone produced is considerably less than that produced from exogenous pregnenolone. Since endogenous progesterone accumulation is almost completely blocked by the addition of aminoglutethimide to the culture medium, it is concluded that this response does represent de novo progesterone synthesis. Neither bovine luteinizing hormone (LH) nor human chorionic gonadotrophin (hCG) affects 3beta-HSD activity in cultured ovarian cells. The ability of the cells to secrete or to further metabolize the progesterone formed is also unaffected. However, both LH and hCG stimulate endogenous progesterone production within one hour of their addition to the culture medium. The stimulation, 2-10 fold in several experiments, can be maintained for at least six days of culture, and is not a result of an increase in the growth rate of the ovarian cells. As would be expected, the stimulation is blocked by the addition of aminoglutethimide to the culture medium. Taken together, these facts suggest that gonadotrophic hormones stimulate progesterone production by ovarian cells specifically by their action at steps prior to the conversion of pregnenolone to progesterone.

The Metabolism of Cholesterol Esters in Rat Adrenal Cell Suspensions

The regulation of cholesterol ester metabolism was studied using cell suspensions of capsular and decapsulated portions of rat adrenal glands. The cell suspensions were prepared using collagenase as a dispersing agent, and incubations were performed in Krebs-Ringer buffer containing 0.1% albumin. The cellular uptake and esterification of exogenously added labeled oleic acid was time and temperature dependent in both capsular and decapsulated cell preparations. Synthetic ACTH caused a decrease in cholesterol ester incorporation with capsular preparations, while no net change was observed in cells from decapsulated adrenal tissue. An increase in oleic acid incorporation into cholesterol esters was observed for both cell preparations when aminoglutethimide was added, particularly in the presence of ACTH. A similar increase was seen with cycloheximide. Corticosterone production, which was stimulated 5–20-fold by ACTH, was reduced to control levels with addition of aminoglutethimide or cycloheximide. When chol...