Column chromatography of steroids on Sephadex LH-20.
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17-Ketosteroids↗
Biomedical subjects
B R Carr
Publications and source records attributed to B R Carr.
Imidazole antimycotics: selective inhibitors of steroid aromatization and progesterone hydroxylation.
Econazole, imazalil, and prochloraz, which have broad spectrum antimycotic activity, are shown to be potent inhibitors of steroid aromatase activity of human placental microsomes. The IC50 values for the inhibition of aromatase activity by econazole, imazalil, miconazole, prochloraz, clotrimazole, ketoconazole, and aminoglutethimide are 0.03, 0.15, 0.6, 0.7, 1.8, 60, and 45 microM, respectively. Econazole and 4-hydroxyandrostenedione also inhibit the steroid aromatase activity of human fetal liver, a finding which suggests that extraplacental aromatase may have many similarities to the placental enzyme. Econazole is a more effective inhibitor of placental aromatization of 19-hydroxyandrostenedione than of androstenedione. This observation is consistent with the competitive nature of the inhibition of aromatase by imidazole antimycotic agents and the reduced affinity of the placental aromatase enzyme for 19-hydroxyandrostenedione compared to androstenedione. The effectiveness of these imidazole antimycotic agents to inhibit the multiple hydroxylations of progesterone which are catalyzed by human fetal adrenal microsomes is also defined. While all of the imidazole antimycotic agents are potent inhibitors of the 16 alpha-, 17 alpha-, and 21-hydroxylations of progesterone, selective inhibitory profiles are apparent. Ketoconazole is a most potent inhibitor of human fetal adrenal progesterone 16 alpha- and 17 alpha-hydroxylases while clotrimazole and imazalil are the most potent inhibitors of progesterone 21-hydroxylase. These results are strongly supportive that imidazole drugs are selective inhibitors not only of steroid aromatase but also of other microsomal steroid hydroxylases.
Adrenal Glands↗
The effects of insulin on 3 beta-hydroxysteroid dehydrogenase expression in human luteinized granulosa cells.
OBJECTIVE: We determined the relative effects of insulin and FSH on progesterone accumulation as well as activity, protein content, and mRNA expression of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in human luteinized granulosa cells. METHODS: Luteinized granulosa cells obtained from women undergoing in vitro fertilization were plated and grown to near confluence and treated with FSH, insulin, or a combination of insulin and FSH. Progesterone production as well as enzyme activity, protein content, and mRNA expression for 3 beta HSD were evaluated. RESULTS: Progesterone production was not affected by insulin alone but increased threefold in the presence of FSH (50 ng/microL) alone. The presence of FSH plus insulin (100 nmol/L) caused a significant increase in progesterone accumulation greater than that of FSH alone. The already high basal levels of 3 beta HSD activity were unaffected by insulin alone but increased 1.7-fold in the presence of FSH. The combination of FSH (50 ng/mL) and insulin (100 nmol/L) increased activity 1.3-fold over FSH alone (P < .02). Insulin (greater than 100 nmol/L) alone increased 3 beta HSD protein content as measured by Western analysis 1.8-2-fold over basal levels, whereas FSH alone increased protein content 2.8-fold, and was further augmented by the addition of insulin in a dose-related fashion up to 3.5-fold over basal levels. Insulin increased 3 beta HSD mRNA twofold over basal levels; FSH alone increased mRNA expression of 3 beta HSD 3.2-fold. In the presence of insulin plus FSH, 3 beta HSD mRNA expression increased 7.6-fold over basal levels. For comparison, insulin also stimulated cytochrome P450 aromatase activity, P450 aromatase protein, and mRNA but to a greater degree than that seen for 3 beta HSD. CONCLUSION: Insulin is a regulator of both 3 beta HSD and aromatase expression in human granulosa cells. Elevated insulin levels could therefore affect steroid production in human granulosa cells and presumably alter the menstrual cycle and fertility.
3-Hydroxysteroid Dehydrogenases↗
17 beta-hydroxysteroid oxidoreductases of human fetal and adult tissues: immunological cross-reactivity with an anti-human placental cytosolic 17 beta-hydroxysteroid oxidoreductase antibody.
To determine whether the immunological determinants of human placental 17 beta-hydroxysteroid oxidoreductase (17 beta-HSOR) were present in 17 beta-HSORs of tissues of the human fetus and adult and of various non-human cells maintained in culture, western immunoblot analysis was conducted by use of a polyclonal antibody directed against determinants of the placental cytosolic enzyme. Tissues and cells were evaluated for the presence of immunocross-reactive proteins with a relative molecular mass (Mr) similar to that of placental 17 beta-HSOR (approximately 34,000). By use of homogenates of human fetal tissues, immunostaining of 17 beta-HSORs of Mr approximately 34 kDa was detected in trophoblast, fetal adrenal neocortex, fetal zone of the adrenal gland, liver, intestine, kidney, brain, lung, skin, heart, spleen, pancreas, chorion laeve, and, occasionally, amnion. Immunostaining at Mr approximately 34 kDa also was demonstrated by use of cytosolic preparations of fetal tissues and, in some cases, by use of unwashed microsomal fractions; this protein was either absent or present in almost undetectable amounts in washed microsomes, except for placenta and fetal brain. Immunostaining at approximately 34 kDa was demonstrated occasionally in decidua of pregnant women by use of homogenates, but was not detected in endometrium or myometrium of non-pregnant women, testis of an adult man, mouse and rat Leydig tumour cells, mouse and rat adrenal tumour cells, and normal bovine adrenocortical cells.
Adult↗
Effect of estradiol on DHEAS production in the human adrenocortical cell line, H295R.
OBJECTIVE: To determine if estradiol regulates DHEA and DHEAS production in a human adrenocortical (H295R) cell line and to determine if this effect is receptor mediated. METHODS: NCI-H295 (H295R) cells were rinsed and placed in phenol red free Dulbecco's Modified Eagle's-F12 medium supplemented with 0.1% charcoal-stripped serum. After 24 hours, cells were rinsed and treated based on experimental design. The effects of estradiol were investigated by: 1) treatment of cells with increasing concentrations of estradiol (300-3000 nmol/L) with or without forskolin (10 mumol/L), 2) treatment of cells with the nonsteroidal synthetic estrogen diethylstilbestrol (DES) (300-3000 nmol/L) with or without forskolin (10 mumol/L), and 3) treatment of cells with an estradiol antagonist (ICI 182, 780) in the presence of estradiol. RESULTS: Estradiol alone increased the basal production of DHEAS in H295R cells in a concentration-dependent manner with a maximal effect at 1000 nmol/L. Forskolin treatment increased the basal production of DHEAS ten-fold. Estradiol also increased the forskolin stimulation of DHEAS production two-fold. In contrast, DES alone or DES in addition to forskolin did not stimulate DHEAS production. Estradiol, in contrast, inhibited H295R adrenal cell production of cortisol whereas DES exhibited a similar inhibition. The estrogen receptor antagonist ICI 182,780 was unable to inhibit the stimulatory effect of estradiol. Finally, estradiol in a concentration-dependent manner suppressed 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity in H295R adrenal cells. CONCLUSION: These experiments support the role of estradiol in regulation DHEAS production by inhibiting 3 beta HSD activity; however, the mechanism appears to require high concentrations of estradiol and appears to be independent of the estrogen receptor.
3-Hydroxysteroid Dehydrogenases↗
Treatment of antibody-associated sperm with media containing high serum content: a prospective trial of fertility involving men with high antisperm antibodies following intrauterine insemination.
PROBLEM: Antisperm antibodies (ASAs) associated with the sperm surface can significantly influence oocyte fertilization. We initiated a prospective trial comparing the effect of serum-medium dilution on ASA binding and/or distribution following ejaculation and on subsequent pregnancy rates following intrauterine insemination (IUI). METHOD: Infertile couples (N = 16) were entered into this prospective randomized protocol where the husband's sperm was at least 50% positive for both IgA and IgG ASAs using the immunobead assay. Couples underwent IUI with washed sperm collected during ejaculation into (a) 10 ml of serum medium made up of 50% maternal serum (antibody negative) in Hepes-buffered medium (SM) or (b) in a sterile cup (DRY). For the following cycle, each couple received the alternate sperm treatment. All patients underwent at least two DRY and two SM collections prior to beginning IUI. Sperm from these collections were analyzed by manual semen analysis, computer-aided semen analysis (CASA), and immunobead testing. All sperm preparations for IUI were analyzed by manual analysis and CASA before IUI. Outcome measures were changes in the distribution and amount of sperm antibody binding, sperm motion parameters, and pregnancy rates following IUI. Statistical analysis was performed using Fisher's exact test. RESULTS: Collection of sperm into SM significantly reduces (P < 0.01) the percentage of antibody-bound sperm (54.8% IgA, 60.0% IgG) versus 83.5% IgA and 87.7% IgG with DRY collection. The distribution or pattern of antibody binding to the sperm also was altered by SM. There was no significant difference between the motility of the sperm following collection with SM or DRY sperm as determined by manual and CASA methods. More importantly, there was no statistically significant difference in the pregnancy rates/treatment cycle following SM (3.1% 32 cycles) or DRY (6.7%, 30 cycles). CONCLUSION: Our data suggests that SM collection alters immunobead detectable ASA binding. Localization of ASA binding sites suggests that head-bound antibodies are influenced by SM treatment with little effect on tail-bound antibodies. In spite of the significant reduction of ASAs present on the sperm following SM treatment, there was no influence on pregnancy rates.
Autoantibodies↗
Regulatory effects of multifunctional cytokines and steroid hormones on apolipoprotein B production by human fetal hepatocytes.
OBJECTIVE: The purpose of the present investigation was to determine the effects of certain multifunctional cytokines (tumor necrosis factor-alpha [TNF-alpha], interleukin-1 beta [IL-1 beta], and IL-6) and steroid hormones (estradiol, testosterone, progesterone, and cortisol) on the production of apolipoprotein B (Apo B) by cultured human fetal hepatocytes. We conducted these experiments because of our recent observations that purified human fetal Kupffer cells produce TNF-alpha, IL-1 beta, and IL-6. METHODS: Human fetal hepatocytes, specifically depleted of hematopoietic precursors and Kupffer cells, were cultured in defined medium. The amounts of Apo B released into the culture medium were measured by a sensitive and specific enzyme-linked immunosorbent assay. RESULTS: Hepatocytes (10(6) cells) produced 82 +/- 16 ng Apo B per 24 hours during days 4-7 in culture. Results demonstrated that treatment of cultured hepatocytes with TNF-alpha maximally inhibited Apo B production by 50% at a half-maximal concentration of 100 pg/mL, whereas IL-1 beta maximally inhibited Apo B production by 80% at a half-maximal dose of 200 pg/mL. Cells exposed to IL-6 produced increased amounts of Apo B, but only after IL-6 was removed from the culture medium. The addition of TNF-alpha, IL-1 beta, or IL-6 did not significantly affect hepatocyte viability. At physiologic concentrations (1 mumol/L), estrogens were able to increase the production of Apo B by 25-65%; however, no positive or negative effect could be demonstrated with dexamethasone, cortisol, testosterone, or progesterone. When using synthetic estrogens such as ethinyl estradiol and mestranol, the stimulatory effect was most pronounced. CONCLUSION: Tumor necrosis factor-alpha and IL-1 beta have an inhibitory effect and estrogens have a stimulatory effect on Apo B production by human fetal hepatocytes in culture. These studies suggest that fetal hepatocytes can produce Apo B and that the synthesis of Apo B is under the control of multifunctional cytokines and steroid hormones.
Apolipoproteins B↗
Steroid cell tumor associated with primary amenorrhea and virilization.
A 22-year-old woman sought medical advice because of primary amenorrhea and virilization that was manifested by facial hirsutism, temporal balding, and clitorimegaly. Plasma steroid levels were determined at the time of initial assessment; androstenedione and testosterone were increased in comparison with normal values. Vaginal ultrasonography revealed the presence of a mass localized to the right ovary. The patient underwent oophorectomy, and pathologic examination of the surgical specimen led to the identification of a steroid cell tumor associated with a polycystic ovary. Tumor steroid-metabolizing enzymes were evaluated in vitro: 17alpha-hydroxylase, 3beta-hydroxysteroid dehydrogenase/ delta5-->4-isomerase, 17beta-hydroxysteroid oxidoreductase, and 5a-reductase, which are required for androgen synthesis, were present in the tumor tissue. Postoperatively, plasma androstenedione and testosterone levels returned to normal. This study demonstrated that the tumor was the source of the increased levels of androgen precursor and androgen in this woman; excess tumor-derived androgen may have been the trigger in the development of the polycystic ovary.
Journal Article↗
Cycle control with desogestrel-containing oral contraceptives--comparison of a monophasic and triphasic regimen.
OBJECTIVE: To examine the effect of mono- and triphasic desogestrel-containing oral contraceptives on cycle control. SETTING: Multicenter, international (U.S. and Canada). PATIENTS AND INTERVENTIONS: About 4,000 women, aged 18-35, healthy, neither pregnant nor lactating, were followed for almost 37,700 cycles of use of a monophasic 150 micrograms desogestrel/30 micrograms ethinyl estradiol OC or Sunday- or day-1 start regimens of a triphasic preparation containing 50/100/150 micrograms desogestrel/35/30/30 micrograms ethinyl estradiol administered on a 7/7/7 schedule. Standard diary methods were used to collect data on breakthrough bleeding, spotting, and absence of withdrawal bleeding. RESULTS: The two formulations demonstrated very low and, in fact, remarkably similar rates of breakthrough bleeding, spotting, and absence of withdrawal bleeding. A statistical analysis of these characteristics of the total study populations, per cycle, revealed no statistically significant differences between the formulations. CONCLUSION: Although the triphasic formulation contains the lowest estrogen dose of any triphasic available, 33% less progestin than the monophasic formulation, it maintains comparable cycle control.
Adult↗
Expression of 11 beta-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) in the human fetal adrenal.
OBJECTIVE: To understand better the steroidogenic capacity of the human fetal adrenal (HFA), we evaluated the expression of 11 beta-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) in the fetal zone and neocortex of the HFA using a specific RNase protection assay. METHODS: Adrenal glands were obtained at the time of elective termination of pregnancy. Whole adrenals (n = 7) were frozen in liquid nitrogen, and subsequently total RNA extraction was performed by tissue homogenization followed by guanidinium/chloroform purification. In addition, RNA was obtained from separated fetal zone (n = 4) and neocortex (n = 4) tissues obtained by dissection. RNase protection assays were then performed using radiolabeled complementary RNA probes generated by T7 RNA polymerase directed against transcripts for CYP11B1, CYP11B2, and actin, the latter of which was used as a control for RNA integrity. Transcripts also were examined using a reverse transcription polymerase chain reaction (RT-PCR) protocol specific for CYP11B1 or CYP11B2. RESULTS: The RNase protection assay was designed to distinguish specific bands that corresponded to CYP11B1 (232 bp), CYP11B2 (262 bp), and actin (221 bp). RNA isolated from whole HFA was observed to have high levels of CYP11B1 transcript, whereas CYP11B2 was not detected. Dissected neocortex and fetal zones were found to contain transcript for CYP11B1 using both the RNase protection assay and RT-PCR analysis. In contrast, using the RNase protection assay, CYP11B2 mRNA was not observed in the RNA from the fetal zone, but after prolonged exposure there was a band corresponding in size to CYP11B2 observed in RNA from the neocortex. Using the more sensitive RT-PCR method, transcript for CYP11B2 was found in both neocortex and fetal zone. CONCLUSION: The HFA expresses low levels of CYP11B2 in accordance with its low production of mineralocorticoid. The expression of CYP11B1 in the fetal zone is intriguing because this enzyme is not necessary for the production of C19 steroids. Definition of the molecular mechanisms controlling expression of the CYP11B genes will be necessary to determine why the HFA differentially expresses these isoenzymes.
Actins↗
Immunohistochemical and biochemical analysis of a human Sertoli-Leydig cell tumor: autonomous steroid production characteristic of ovarian theca cells.
OBJECTIVE: To ascertain the steroidogenic profile and location of steroidogenic enzymes in a steroid-secreting Sertoli-Leydig cell tumor of the ovary. METHODS: Steroid levels from peripheral, left ovarian (tumor), and right ovarian venous blood were measured. Tumor tissue was examined for the steroidogenic enzymes 17 alpha-hydroxylase (P450c17) and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) by immunohistochemistry. Tumor cells were isolated and incubated in serum-free media. Thereafter, media were analyzed for steroid production, steroidogenic response to effectors, and metabolism of radiolabeled pregnenolone and androstenedione. RESULTS: Levels of C19 steroids and 17-hydroxyprogesterone (17OHP) were elevated in peripheral blood. The majority (80%) of steroids in serum from the left ovarian vein (tumor) were C19 steroids (dehydroepiandrosterone [DHEA], 45%; androstenedione, 27%, testosterone, 7%, with 13% 17OHP, 7% progesterone, and less than 1% estradiol (E2). Immunoreactivity for both P450c17 and 3 beta HSD was identified in clusters of large cells surrounded by nonimmunoreactive cells composing cord-like structures. Of the steroids that accumulated in the incubation medium of unstimulated, freshly isolated tumor cells, 84% were C19 steroids (DHEA, 44%; androstenedione, 36%; testosterone 2%, with 16% 17OHP and less than 1% progesterone and E2. Basal steroid production was not stimulated by LH or FSH. However, treatment with forskolin (10 mumol/L), dibutyryl cAMP (1 mmol/L), or steroid precursors (22-hydroxycholesterol, 1 mumol/L; pregnenolone, 1 mumol/L) increased the production of all steroids measured. Forskolin treatment increased androstenedione (fivefold), DHEA (tenfold), and 17OHP (40-fold) compared with basal levels. Incubation of freshly isolated cells with [3H]pregnenolone demonstrated the ability of these cells to metabolize this C21 steroid precursor to androstenedione, DHEA, and 17OHP. However, [3H]androstenedione was not readily metabolized by these cells to either estrone or testosterone. CONCLUSIONS: The steroidogenic properties of a steroid hormone-producing tumor were described. Cells isolated from this tumor produced steroids similar to those secreted by ovarian theca cells. These properties suggest that certain ovarian steroidogenic tumor cells may be an appropriate model for ovarian theca cells and could be used to develop steroid-secreting cell lines.
3-Hydroxysteroid Dehydrogenases↗