Showing papers on "Pregnenolone published in 1981"

Cytochrome P-450 Monooxygenase Activities in Human and Rat Liver Microsomes

Abstract: Microsomes were prepared from human livers obtained from renal donors of various ages and both sexes. Their drug-metabolizing capacity was measured and compared to that of rat liver microsomes. The following parameters were investigated: cytochrome P-450, cytochrome B5, NADPH-cytochrome c reductase, epoxide hydrolase, aryl hydrocarbon hydroxylase, benzphetamine N-demethylase, p-nitroanisole-O-demethylase, ethoxycoumarin-O-deethylase, steroid-16 alpha-hydroxylase. In addition, the metabolism of benzo(a)pyrene, progesterone, pregnenolone, testosterone, dehydroepiandrosterone and estradiol was studied in detail in vitro. The inhibitory effect of metyrapone and alpha-naphthoflavone on 7-ethoxycoumarin-O-deethylase was measured. The microsomal proteins of both species were separated by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. The following conclusions were drawn from the results obtained. Human liver microsomes can be stored under optimal conditions for the measurement of a large variety of enzymic activities. Human liver microsomes are able to metabolize the various xenobiotics used as substrates with a rate similar to that of female rat liver microsomes. No sex-linked difference in enzymic activity was observed in human microsomes. Significant differences in benzo(a)pyrene and steroid metabolism were registered when human and rat liver microsomes were compared. The monooxygenase activities, the sensitivity to in vitro alpha-naphthoflavone and metyrapone, the results of steroid metabolism, and slab gel electrophoresis are strong indications for multiplicity of human liver cytochrome P-450.

Age-related changes in endogenous steroids of human fetal testis during early and midpregnancy.

Abstract: The steroidogenic activity of human fetal testes during early and midgestation was monitored by analyzing 58 individual fetal testes (aged 6–20 weeks of pregnancy) for endogenous pregnenolone (P5), progesterone, 17-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, testosterone (T) and estradiol. A clear increase in testicular steroid concentrations, especially in those of T and other 3-keto-4-ene steroids, occurred between 8–11 weeks of gestation and reached maximum between 11–14 weeks. The three steroids present in highest concentrations were P5 androstenedione, and T (maximum concentrations, 1.9–2.7 ng/mg wet tissue). The levels of all of the C-19 steroids measured decreased clearly between weeks 14–20 of gestation, whereas those of the C-21 steroids, P5, progesterone, and 17-hydroxyprogesterone, remained relatively high. Our results suggest that the metabolic reactions converting P5 to androgens are activated in the human fetal testis within a short time range between 8–11 weeks of gestatio...

The Influence of Calmodulin on Steroid Synthesis in Ley dig Cells from Rat Testis

Abstract: Two approaches were used to study the possible role of calmodulin in the regulation of synthesis of testosterone by Leydig cells: trifluoperazine was used as an inhibitor of calmodulin and liposomes were used to deliver calmodulin into the cells. The inhibitor prevented the expected responses of Leydig cells to LH and to cAMP. First the increase in synthesis of testosterone produced when these agents are added to Leydig cells was inhibited by the drug. Second, increased transport of cholesterol to mitochondria produced by LH and cAMP was inhibited by trifluoperazine. Third, increased side-chain cleavage of cholesterol (cholesterol leads to pregnenolone) produced by these agents in isolated mitochondria was also inhibited by the drug. When Leydig cells were incubated with liposomes containing calmodulin, production of testosterone, transport of cholesterol to mitochondria, and side-chain cleavage of cholesterol were all stimulated. The effect of calmodulin is greater if Ca2+ is added before incorporation into liposomes than if calmodulin and Ca2+ are introduced into the Leydig cells from separate liposomes. Stimulation of testosterone synthesis does not occur if calmodulin is dialyzed against EGTA, if calmodulin with excess anticalmodulin is present in the liposomes, if either calmodulin or Ca2+ is added to the medium (no liposomes), or if Ca2+ alone is present in liposomes. These observations suggest that calmodulin is involved in regulating the transport of cholesterol to mitochondria, a process that is stimulated by LH and cAMP and one that may account for the increased steroid synthesis produced by these agents.

Characterization of steroid production in cultured human choriocarcinoma cells

Abstract: Progesterone is the major steroid synthesized by the JEG-3, BeWo, and JAR cell lines of choriocarcinoma. A lesser amount of pregnenolone is produced. The 17alpha-hydroxy derivatives of these steroids are only minimally present in three lines. The addition of fetal calf serum to the culture medium modestly increases the synthesis of these steroids, but increases the quantity of 17beta-estradiol produced by 30- to 90-fold. The addition of dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, androstenediol, and testosterone was shown to stimulate 17beta-estradiol synthesis. There is a clear dose-response relationship between the amount of testosterone added and the quantity of 17beta-estradiol produced. These results indicate that 3beta-hydroxysteroid dehydrogenase-isomerase, 17beta-ol dehydrogenase, and aromatase are active in cultured choriocarcinoma cells, whereas 17beta-hydroxylase and 17-20 desmolase do not appear to be functional in these cells. It is concluded that the stereoidogenic capabilities of choriocarcinoma cells in culture are similar to those of the in vivo placenta and support their use as an experimental model of placental steroidogenesis.

Inhibition of Testicular Microsomal Cytochrome P-450(17α-Hydroxylase/C-17,20-Lyase) by Estrogens*

Abstract: Highly purified cytochrome P-450 from neonatal pig testicular rnicrosomes is capable of catalyzing both 17αhydroxylation and C-17,20-lyase activity. Estradiol was found to inhibit both activities of the purified enzyme with Δ 4 and with Δ 5 substrates (progesterone, pregnenolone, and the corresponding 17α-hydroxysteroids). For the Δ 4 series, inhibition of lyase is competitive and that of 17α-hydroxylase is noncompetitive; for the Δ 5 series, inhibition was noncompetitive for both activities. Ki values for lyase activity were determined from enzyme kinetics (5.0 μM for the Δ 4 substrate and 20 μM for the Δ 55 substrate). Estradiol produces a typical type I spectral shift with the pure enzyme (Ks = 3.0 μ where Ks is the concentration of steroid required to give half maximal spectral shift), so that Ki values were also determined directly from binding studies by using substrate-induced difference spectroscopy. Fifty per cent inhibition of the maximal spectral shift induced by the 17α-hydroxysubstrates (Ki) ...

Mechanism by which 17β-Estradiol Inhibits Ovarian Androgen Production in the Rat*

Abstract: The mechanism by which 17β-estradiol inhibits ovarian androgen biosynthesis was investigated. Immature 25-day-old rats were treated for 2 days with estradiol, after which whole ovaries were dispersed, and gonadotropin binding and cAMP and steroid hormone production were examined. When dispersed cells from untreated control ovaries were incubated with hCG (100 ng/ml), there were marked increases (10-fold) in steroid production, with the major steroids being progesterone and androstenedione. The stimulation of steroidogenesis by hCG was dose-related (ED50 = 100 pg/ml hCG). After 2 days of estradiol treatment, the maximum hCG stimulation of androstenedione, testosterone, 17α-hydroxypregnenolone, and 17α-hydroxyprogesterone production by ovarian cells was inhibited by 90%; pregnenolone production was unchanged, while progesterone production was increased by 30%. Time course studies showed that the stimulatory effect of hCG on androgen production was maximally inhibited (90%) after 12 h of estradiol treatment....

The Control of Steroidogenesis by Human Fetal Adrenal Cells in Tissue Culture. I. Responses to Adrenocorticotropin

Abstract: A technique of monolayer tissue culture of human fetal adrenal cells was developed in order to study steroidogenic responses to factors such as ACTH. The daily production of 12 steroids [pregnenolone, 17-hydroxy pregnenolone, dehydroepiandrosterone (DHA), DHA sulfate, progesterone, 17-hy-droxyprogesterone, aridrostenedione, testosterone, corticoster-one, 11-desoxycortisol, cortisol, and aldosterone) was measured by RIA. Initially, fresh’fetal adrenal cells produced DHA, DHA sulfate, 17-hydroxypregnenolone, and small amounts of cortisol, but in _he absence of ACTH, the production of all steroids declined during culture to low levels. The addition of physiological amounts (1–104 pg/ml) of either αACTH-(l–24) or αACTH-(1–39) or coculture with fetal pituitary cells elicited a progressive rise in steroid production during the first 4–6 days of incubation. The lowest ACTH doses elicited a proportionately greater adrenal androgen response (as reflected in the DHA to cortisol ratio), but with increasing ACTH dosa...

Functional differences between the outer and inner zones of the guinea pig adrenal cortex

Abstract: The guinea pig adrenal cortex is grossly composed of two regions: an outer, yellow zone and an inner, brown zone. These zones,which represent 33% and 66% of the total adrenocortical volume,respectively,can be separated by blunt dissection. It has been previously reported that specific pregnenolone and pregnenolone sulfate binding proteins are present in the high speed supernatant fraction (cytosol) prepared from the whole adrenal cortex of the. guinea pig. However, when cytosol was prepared from the separate outer and inner cortical zones, it was found that the steroid-binding proteins were concentrated in the inner zone. This correlated with the level of pregnenolone which was significantly greater in the cytosol of the inner zone where greater than 50% was found to be bound. In contrast, the concentration of cortisol was 30 times greater in the cytosol of the outer cortical zone and less than 4% was found to be bound. These data suggest that cortisol is produced primarily in the outer cortical zone, a e...

Steroid Secretion by Sexually Immature Rat and Rabbit Testes Perfused in Vitro

Abstract: Puberty in the mammal signals a dramatic change in testicular function. Previous studies of steroid production by pubertal rat and rabbit testes have been incomplete and the results have not been comparable because of the variety of techniques used. The purpose of the present experiments was to quantify and compare the in vitro testicular steroid secretion profiles of rats and rabbits at the same pubertal stage as defined by spermatogenic development: near the completion of meiosis. The secretion rates of testosterone, its 5α-reduced metabolites (dihydrotestosterone, 3α-androstanediol, 3β-androstanediol), and testosterone biosynthetic intermediates (pregnenolone, 17α-hydroxypregnenolone, dehydroepiandrosterone, androstenediol, progesterone, 17α-hydroxyprogesterone, androstenedione) were measured in order to elucidate testicular steroidogenic capacity. Steroids in the venous effluent of testes perfused in vitro were extracted, isolated by thin layer and Celite-column chromatography, and subsequently quanti...

In vitro oestrogen synthesis by bovine placenta during pregnancy and induced parturition

Abstract: Foetal (cotyledon) placental tissue was collected from 7 pregnant cows and from 8 cows immediately after delivery of the foetus during parturition induced by luteal removal at 250 or 270 days of gestation. These tissues were incubated with [3H]androstenedione (A), [3H]17 alpha-hydroxyprogesterone (17 alpha HP), or [3H]pregnenolone (P5) to examine potential oestrogen precursors in the mid to late pregnant cow. Percentage conversion to unconjugated and conjugated oestrone (Oe1) and oestradiol (Oe2) was measured for each tissue with the various substrates. Unconjugated oestrogens predominated over conjugated (P less than 0.001), and Oe1 over Oe2 (P less than 0.001) in all incubations. There was no difference in conversion of A between pre- and post-partum foetal tissues. There was a significant elevation in conversion of P5 in post-partum foetal tissue (P less than 0.05). These data demonstrate a) placental aromatase activity does not appear to increase as pregnancy progresses towards parturition, b) the bovine placenta is able to convert C21 steroids to oestrogens, and c) induction of enzymes for converting pregnenolone or related substrates to oestrogens may be involved in the pre-partum rise in oestrogen levels.

Enzyme-bound sterols of bovine adrenocortical cytochrome P-450scc.

Abstract: Bovine adrenocortical cytochrome P-450 capable of cleaving the side chain of cholesterol was purified to homogeneity by the method of Suhara et al. [Suhara, K., Gomi, T., Sato, H., Itagaki, E., Takemori, S., & Katagiri, M. (1978) Arch. Biochem Biophys. 190, 290]. The substrate-bound form of the enzyme preparation was shown to contain in addition to cholesterol (1.2-3.0 mol/mol of P-450) 0.4 mol of (22R)-22-hydroxycholesterol, 0.1 mol of (20R,22R)-20,22-dihydroxycholesterol, and a trace amount (0.005 mol) of (20S)-20-hydroxycholesterol per mol of P-450scc. This relatively large concentration of (22R)-22-hydroxycholesterol is in accord with the hypothesis that the major pathway leading to side-chain cleavage proceeds through initial hydroxylation at the 22 position. The presence of these sterols as native constituents of cytochrome P-450scc supports their role as enzyme-bound intermediates in the biosynthesis of pregnenolone from cholesterol.

Lutropin-dependent protein phosphorylation and steroidogenesis in rat tumour Leydig cells

Abstract: Tumour Leydig cells have been incubated in the presence or absence of lutropin (luteinizing hormone, ;LH'). Stimulation of cells with lutropin (1000ng/ml) in the presence of 1-methyl-3-isobutylxanthine (0.25mm) resulted in increased steroid production and increased protein phosphorylation. When pregnenolone metabolism was inhibited, basal pregnenolone production was 26.9+/-7.4ng/60min per 10(6) cells; stimulated production was 156.1+/-39.5ng/60min per 10(6) cells (means+/-s.d., n=4). Lutropin-dependent phosphorylated proteins of molecular mass 17000, 22000, 24000, 33000 and 57000Da were detected. A significant increase of [(32)P]P(i) incorporation into these phosphorylated proteins was observed concomitant with the increased pregnenolone production. The occurrence of the phosphoproteins in nuclei, mitochondria and postmitochondrial-supernatant was investigated. The 17000Da phosphoprotein was found in the nuclear fraction, whereas the 22000, 24000, 33000 and 57000Da phosphoproteins were localized in the postmitochondrial-supernatant fraction. Of the cholesterol-side-chain-cleavage activity, 80.3+/-6.1% (mean+/-s.d., n=5) was present in the mitochondrial fraction isolated from tumour Leydig cells, and this activity was 2.5-fold increased when cells had been preincubated with lutropin/1-methyl-3-isobutylxanthine (basal production: 194.6+/-28.6ng/30min per mg of protein; lutropinstimulated production: 498.8+/-91.5ng/30min per mg of protein; means+/-s.d., n=3). The similarities in the kinetics of the phosphorylation of proteins and the pregnenolone production after addition of lutropin/1-methyl-3-isobutylxanthine indicate that the phosphoproteins could be involved in the lutropin-dependent increase in steroidogenesis in tumour Leydig cells. It remains to be demonstrated, however, to what extent the phosphoproteins outside the mitochondria can influence the cholesterol-side-chain-cleavage activity inside the mitochondria.

Effects of Estrogen Treatment on Human Testicular Unconjugated Steroid and Steroid Sulfate Production in Vivo

Abstract: To analyze the relationships between human testicular unconjugated and sulfate-conjugated steroids, we investigated the effects of estradiol [polyestradiol phosphate (E2)] and a synthetic estrogen [diethylstilbestrol diphosphate (DESDP)] on testicular, spermatic venous, and peripheral venous serum concentrations of these compounds. Six unconjugated steroids and 4 steroid sulfates were measured by specific RIAs after chromatography in 27 prostatic carcinoma patients without preceding endocrine therapy and in 29 patients castrated after E2 or DESDP treatment. E2 treatment decreased testicular concentrations of pregnenolone, testosterone (T), and 5 a-dihydrotestosterone (DHT). Testicular androstenedione concentrations were also decreased, whereas progesterone concentrations increased, and there were decreased testicular concentrations of dehydroepiandrosterone sulfate (DHEA-SO4) and 5-androstene-3β,17β-diol sulfate. Spermatic venous concentrations of 17a-hydroxyprogesterone (17-OHP), T, and DHT as well as th...

Steroid Secretion by Masculinizing and “Feminizing” Hilus Cell Tumors

Abstract: The clinical course, histology, and steroid secretion observed in two patients with hilus cell tumors are presented. One patient had signs of virilism and the other had estrogenic signs only. Steroid secretion was examined by measuring peripheral and ovarian venous gradients and pre-and postoperative levels of hormones to explain the profound differences in the biological effects exerted by the neoplasms. In the patient with virilism, the tumor's major secretory product was testosterone (T), and the dominant biosynthetic pathway was pregnenolone (Pe) → 17-hydroxypregnenolone → 17-hydroxyprogesterone → androstenedione (Δ) → T. In the patient with estrogenic signs, the major secretory product was Δ, derived from a similar pathway of pregnenolone → 17-hydroxypregnenolone → 17-hydroxyprogesterone → Δ Circulating estrone and estradiol levels were elevated, but the tumor showed limited aromatase activity, as reflected by 60-to 1500-fold larger peripheral-ovarian venous gradients of Δ and T than estrone and estr...

Functional and Structural Relationships in Steroidogenesis in Vitro by Human Corpora Lutea during Development and Regression

Abstract: To investigate the steroidogenic function of human corpora lutea, during the menstrual cycle, slices of seven corpora lutea obtained at various stages in the luteal phase were incubated with [1-14C]acetate; four of them were incubated with [4-14C]pregnenolone simultaneously Incorporation or conversion of 14C radioactivity into progestins, androgens, and estrogens was assessed by a reverse dilution technique with recrystallization to constant specific activity Distinct differences in function and morphology were observed before and after the completion of corpus luteum formation In developing corpora lutea, 1) a marked increase in progesterone formation from [14C]acetate, 2) a transient increase in androgen formation, including dehydroepiandrosterone, with a concomitant decrease in estrogen formation from the two precursors, and 3) degeneration, followed by disappearance of thecal cells, were observed In mature and regressing corpora lutea, 1) a drastic decrease in progesterone formation from [14C]acetate, 2) an increase in estrogen in contrast to a decrease in androgen formation from the two precursors, 3) a clear differentiation of theca lutein cells, and 4) a more relatively pronounced transformation of pregnenolone to estrogen in regressing than in mature corpora lutea were found We conclude that marked qualitative and quantitative changes in steroidogenic function are closely related to structural changes during corpus luteal development and regression