Patterns of 3β-hydroxysteroid dehydrogenaseΔ5−4isomerase activity in the rhesus monkey placenta and fetal adrenal

3β-Hydroxysteroid $\text{dehydrogenase}\text{Δ}{}^{\mathrm{5?4}}\text{isomerase}$ activity (3Δ-HSDH) was examined in the rhesus monkey ($\text{Macaca mulatta}$) placenta and fetal adrenal at 135 and 155–162 days of gestation. Activity was evaluated in microsomes by the conversion of [³H]pregnenolone to [³H]progesterone. There was a 7-fold increase in enzyme activity in the whole adrenal (minus medulla) between the two stages of development. Combining data from both periods, enzyme activity was greater in the outer than in the inner region of the adrenal. No stage-dependent change in placental activity was evident. The temporal patterns in 3β-HSDH activity are consistent with corticoid and progesterone patterns in the circulation. Thus, the level of 3β-HSDH activity may be rate limiting in both the fetal adrenal and placenta.Enzyme activity was assessed in incubations which included unex-tracted, heat-treated, 100,000 g tissue supernatants. In both placental and adrenal incubations, competitive inhibition was noted. Ethyl ether extracts of 100,000 g tissue supernatants also inhibited 3β-HSDH in the respective tissues. GLC analysis of these extracts revealed the presence of putative dehydroepiandrosterone. Hormone levels and the nature of the inhibition that were observed are compatible with the conclusion that dehydroepiandrosterone can inhibit the conversion of pregnenolone to progesterone $\text{in vivo}$. The physiological importance of this remains to be determined.     

Effects of adrenal steroid activator protein on the conversion of various 20- and 22-hydroxycholesterols to pregnenolone by adrenal mitochondrial enzymes

A heat-stable protein activator from bovine adrenal cortex mitochondria stimulates the conversion of cholesterol to pregnenolone in crude extracts of adrenal mitochondria, and resembles in some of its properties, the sterol carrier protein of liver (Kan $\text{et}\text{al}$. $\text{Biochem}$. $\text{Biophys}$. $\text{Res}$. $\text{Commun}$. $\text{48}$, 423–429, 1972). We have shown that activator preparations also stimulate highly purified adrenal enzyme preparations comprising four components: cytochrome P-450 specific for side chain cleavage, adrenodoxin, adrenodoxin reductase, and an NADPH-generating system. Furthermore, this activator stimulates the conversion not only of cholesterol, but also of (20S)-20-hydroxycholesterol, (22$\text{R}$)-22-hydroxycholesterol, and (20$\text{R}$, 22$\text{R}$)-20,22-dihydroxycholesterol to pregnenolone. Our findings provide additional evidence that the steroid-activator complexes are the substrates for the side chain cleavage enzyme and that the monohydroxy and dihydroxycholesterols are true intermediates in the conversion of cholesterol to pregnenolone by bovine adrenal cortex mitochondria.     

Inhibition of testosterone biosynthesis by ethanol: relation to the pregnenolone-to-testosterone pathway

The concentrations of metabolites in the pregnenolone → testosterone pathway were determined in freezestopped testes in control rats and during ethanol intoxication (2 h after injection of 1.5 $\text{g ethanol}\text{kg}$ body wt). Ethanol lowered the mean testicular concentrations of testosterone (by 63–74%), androstenedione (49–81%), 17-hydroxyprogesterone (60–76%), progesterone (29–67%) and pregnenolone (12–25%). 4-Methylpyrazole had no effect on the ethanol-induced changes. The present results reveal no inhibition at the 17-hydroxyprogesterone → androstenedione → testosterone steps, but do not exclude inhibition before the step yielding pregnenolone and at the pregnenolone → progesterone → 17-hydroxyprogesterone steps.     

Contribution of microsomal cytochrome b5 electron transport system coupled with Δ5-3β-hydroxysteroid dehydrogenase to androgen synthesis from progesterone

Cytochromes P-450 and $\text{b}$₅ were observed in the microsomal fraction of interstitial tissue of rat testes. Microsomal cytochrome $\text{b}$₅ was reduced by the NADH coupled with the activities of Δ⁵-3β-hydroxysteroid dehydrogenase with Δ⁵-Δ⁴ isomerase through conversion of pregnenolone to progesterone. Activities of NADPH-supported 17α-hydroxylase and C-17-C-20 lyase which converted progesterone to androstenedione were stimulated by either the presence of NADH or the oxidative reaction by the dehydrogenase upon Δ⁵-3β-hydroxysteroids. Androstenedione production enhanced by the reaction of the dehydrogenase was decreased by addition of the antibody against NADH-cytochrome $\text{b}$₅ reductase which was purified from rat hepatic microsomes, suggesting the active participation of cytochrome $\text{b}$₅ in the androgen synthesis.     

Effect of estradiol-17β on the conversion of pregnenolone to progesterone in human term placenta incubated in vitro

The effect of different doses of estradiol-17β (E₂) on the netabolic pregnenolone to progesterone pathway in fragments of human term placenta incubated $\text{in vitro}$ was studied. Doses considered as being physiological of 0.09 and 0.9 μM had a stimulatory effect on the conversion (p < 0.008 to 0.0l6). However, a supraphysiological dose of 45 μM showed an inhibitory activity related to the maximal stimulation (p < 0.03). A dose of 0.9 μM E₂ favoured the accumulation of (³H)-progesterone in the tissue (p < 0.05). These results suggest that E₂ may regulate the synthesis of progesterone in human term placenta.     

Androgen regulation of progestin biosynthetic enzymes in FSH-treated rat granulosa cells in vitro

The influence of androgens on the FSH modulation of progestin biosynthetic enzymes was studied $\text{in vitro}$. Granulosa cells obtained from immature, hypophysectomized, estrogen-treated rats were cultured for 3 days in a serum-free medium containing FSH (20 ng/ml) with or without increasing concentrations (10^?9?10^?6 M) of 17β-hydroxy-5α-androstan-3-one (dihydrotestosterone; DHT), 5α-androstane-3α, 17β-diol (3α-diol), or the synthetic androgen 17β-hydroxy-17-methyl-4,9,11-estratrien-3-one (methyltrienolone; R1881). FSH treatment increased progesterone and 20α-hydroxy-4-pregnen-3-one(20α-OH-P) production by 10.2- and 11-fold, respectively. Concurrent androgen treatment augmented FSH-stimulated progesterone and 20α-OH-P production in a dose-related manner (R1881 > 3α-diol > DHT). In the presence of an inhibitor of 3β-hydroxysteroid dehydrogenase (3β-HSD), the FSH-stimulated pregnenolone (3β-hydroxy-5-pregnen-20-one) production (a 20-fold increase) was further enhanced by co-treatment with R1881, 3α-diol or DHT. Furthermore, FSH treatment increased 4.4-fold the activity of 3β-HSD, which converts pregnenolone to progesterone. This stimulatory action of FSH was further augmented by concurrent androgen treatment. In contrast, androgen treatment did not affect FSH-stimulated activity of a progesterone breakdown enzyme, 20α-hydroxysteroid dehydrogenase(20α-HSD). These results demonstrate that the augmenting effect of androgens upon FSH-stimulated progesterone biosynthesis is not due to changes in the conversion of progesterone to 20α-OH-P, but involves an enhancing action upon 3β-HSDΔ⁵, Δ⁴-isomerase complexes and additional enzymes prior to pregnenolone biosynthesis.     

Regulation of steroidogenesis in the ovine corpus luteum

To examine the factor affecting LH-induced progesterone production $\text{in}$$\text{vitro}$ in ovine luteal slices, an experimental procedure was employed wherein each slice served as its own control. The role of microfilaments in steroidogenesis was studied in luteal slices treated with cytochalasin B (an inhibitor of microfilament function). Cytochalasin B treatment resulted in significant reduction of progesterone production by luteal slices in response to LH and the addition of serum to the medium did not alter this effect. The ability of luteal slices to respond to LH with increased progesterone secretion was restored when cytochalasin B was removed from the medium. Further studies indicated that inhibition of LH-induced progesterone production by treatment with cytochalasin B was not a result of a change in: 1) cyclic adenosine 3'-5'-monophosphate production in response to LH; 2) mitochondrial membrane permeability to cholesterol; or 3) activity of 3β-hydroxysteroid dehydrogenase, Δ⁵,Δ⁴-isomerase enzyme complex.The possibility existed that microfilaments were necessary for cholesterol transport to mitochondria in response to LH stimulation. However, mitochondrial cholesterol content was unchanged in response to LH in the presence or absence of aminoglutethimide (an inhibitor of cholesterol side-chain cleavage enzyme activity) as determined by uptake of ³H-cholesterol or total content determined by gas-liguid chromatography. Further, treatment with cytochalasin B had no effect on mitochondrial cholesterol content. These results suggest a role for microfilaments in LH-induced progesterone production at a point prior to the conversion of cholesterol to pregnenolone.     

Isolated Sertoli cells from immature rats produce 20α-hydroxy-pregn-4-en-3-one from progesterone and 3β,20α-dihydroxy-5α-pregnane from pregnenolone

Sertoli cells from 17 day old rats convert progesterone to 20α-hydroxy-pregn-4-en-3-one and pregnenolone to 3β,20α-dihydroxy-5α-pregnane after 72 hours $\text{in vitro}$. The metabolites were identified by several systems of thin layer and gas chromatography, derivative formation and crystallization with authentic steroids. The production of 20α-hydroxy-pregn-4-en-3-one and 3β,20α-dihydroxy-5α-pregnane amounted to 1380 and 740 pmoles/h/mg protein which can account for the total amounts of these steroids reported in the testis. It is the first direct evidence that Sertoli cells can metabolize progesterone and pregnenolone and suggests that Sertoli cells contain the major, if not the only, amounts of 20α-hydroxysteroid dehydrogenase in the immature rat testis.     

3β-Hydroxysteroid dehydrogenaseΔ5−4isomerase activity in the rhesus monkey placenta and fetal adrenal,testis and ovary during late gestation

3β-Hydroxysteroid $\text{dehydrogenase}\text{Δ}{}^{\mathrm{5?4}}\text{isomerase}$ (3β-HSDH) was measured in the rhesus monkey ($\text{Macaca mulatta}$) placenta, fetal adrenal (whole organ minus medulla), testis and ovary during late gestation (Days 145–162). Activities were evaluated from the conversion of [³H]-pregnenolone to [³H]progesterone. The maximum enzyme velocity (V_m) in adrenal microsomes (100,000 g pellet) was significantly higher (146 nmoles progesterone/h x mg^?1protein) than in microsomes from the other tissues. Testicular V_m was greater than either ovarian or placental V_m which were not different from one another (11.5 versus 1.9, 1.2 nmoles progesterone/h x mg^?1protein, respectively). Apparent Michaelis-Menten constants in the adrenal, placenta, testis and ovary averaged 1.8,2.5,0.27 and 0.16 μM, respectively. In some cases, substrate inhibition was noted. Estimated dissociation constants for pregnenolone were 2.3 μM (adrenal), 2.1 μM (placenta), 0.74 μM (testis) and 0.13 μM (ovary). 3β-HSDH was less active in a crude mitochondrial preparation from the fetal adrenal (10,000 g pellet) than in microsomes, whereas activity in the placenta and testis appeared to be equally distributed between mitochrondria and microsomes.Rate measurements were consistent with the apparent potentials of these organs to synthesize their characteristic hormones. Thus, 3β-HSDH activity may be an important rate determining step in hormone synthesis. The importance of substrate inhibition in progesterone formation remains to be assessed.     

Steroid biosynthesis by reptilian adrenals

Incubations of whole homogenates of. the tiju lizard ($\text{Tupinambis sp}$.) adrenals tissue were carried out using ¹⁴C-labelled progesterone^1*, pregnenolone and cholesterol. ¹⁴C-progesterone was metabolized to labelled 18-hydroxycorticosterone, aldosterone, corticosterone and 11-deoxycorticosterone. Identical metabolites plus ¹⁴C-progesterone were obtained from pregnenolone. Cholesterol-4-¹⁴C was transformed into products similar to those obtained from progesterone. In all these studies the elaboration of cortisol or any other 17-hydroxylated steroids could not be demonstrated. In another set of experiments, whole homogenate preparations from adrenals of the green lizard ($\text{lacerta viridis}$) were incubated with ¹⁴C-labelled androstenedione and testosterone. Ahdrostenedione was converted to testosterone and 11β-hydroxyandrostenedione. Testosterone was metabolized to 11β-hydroxyandrostenedione and androstenedione. The results indicate that the $\text{in vitro}$ transformation of C-27 or C-21 radioactive substrate by lizard adrenals is similar to the other reptiles studied. However, it appears to possess 17β-hydroxysteroid oxido-reductase, though the adrenal tissue itself lacks 17α-hydroxylase activity.     

Inhibition of bovine adrenocortical cytochrome P-450scc by 3,3'-dimethoxybenzidine

The effect of 3,3'-dimethoxybenzidine ($\text{o}$-dianisidine) on the conversion of cholesterol to pregnenolone was investigated in a reconstituted side chain cleavage system using enzymes purified from bovine adrenal cortex; d-$\text{p}$-aminoglutethimide was also assayed under similar conditions for comparison. 3,3'-Dimethoxybenzidine was found to be a potent inhibitor of pregnenolone formation, causing 50% inhibition at a concentration of 1.5 μM when using 70 μM cholesterol — this dose is approximately one fourth that required of 3-methoxybenzidine and one twentieth that required of benzidine for equal inhibition. In the same system, d-$\text{p}$-aminoglutethimide exhibited an I₅₀ value of about 55 μM. No effects of 3,3'-dimetoxybenzidine on adrenodoxin reductase or adrenodoxin activities could be detected, and inhibition of side chain cleavage could be relieved by dilution suggesting that the inhibitor acts by reversibly binding to cytochrome P-450scc.     

Progestin metabolism in the rhesus monkey corpus luteum

For the purpose of describing the pathway by which estrogens are synthesized in the rhesus monkey ($\text{Macaca}$$\text{mulatta}$) corpus luteum (CL), CL were obtained during the midluteal phase of the menstrual cycle and fragments incubated with equimolar amounts of [7-³H]pregnenolone plus [4-¹⁴C]progesterone. Metabolites including ³H-progesterone, ³H, ¹⁴C-20α-dihydroprogesterone, ³H, ¹⁴C-17-hydroxyprogesterone, ³H-estrone and ³H-estradiol-17β appeared in the medium during the first 20 minutes of incubation, ³H, ¹⁴C-Androstenedione was not consistently noted until after 60 minutes. Despite the fact that the ¹⁴C/³H-17-hydroxyprogesterone ratio quickly approached a constant value in the medium, ¹⁴C-estrogens were not detected in the medium or tissue fragments suggesting that progesterone was not a principal precursor for estrogen synthesis. As evidenced by the observation that the ¹⁴C/³H-progesterone ratio was significantly higher in luteal fragments than the 17-hydroxyprogesterone ratio, 17-hydroxyprogesterone appeared to be synthesized from pregnenolone both by way of progesterone and by another route which did not include progesterone. C₂₁- and C₁₈-Steroids were more concentrated in tissue fragments after 120 minutes of incubation than in the medium indicating that these steroids were sequestered by luteal tissue.     

Identification and quantification of unconjugated neutral steroids in adrenal and liver tissue of early and mid-term human fetuses

In order to study further the metabolism of neutral steroids in human fetal adrenal and liver tissue the fractions of unconjugated neutral steroids isolated from these tissues were analyzed by gas-liquid chromatography and gas chromatography — mass spectrometry. In the adrenals, pregnenolone and 17-hydroxypregnenolone, but no corticoids, were detected. In the liver, pregnenolone, 3α-hydroxy-5β-pregnan-20-one, 5β-pregnane-3α, 20α-diol and 3β, 16α-dihydroxy-5β-pregnan-20-one were found. Thus, all the free steroids detected were C₂₁ compounds. From these results and those obtained earlier by the analysis of the sulfate-conjugated steroids present in these tissues it is concluded that in the fetal adrenals $\text{in situ}$ both sulfated and unconjugated steroids are actively metabolized. Regarding the liver it is obvious that the conjugated metabolites of progesterone are rapidly eliminated from this tissue. Here, pregnenolone is present both in the free and sulfate conjugated form, whereas its metabolites are found only as sulfate conjugates.     

De novo synthesis of steroids (from acetate) by isolated rat Sertoli cells.

Sertoli cells from 17 day old rats were shown to convert [¹⁴C]acetate to [¹⁴C]-labelled cholesterol, pregnenolone and 17α-hydroxypregnenolone$\text{in}$$\text{vitro}$. Identification was by several systems of thin layer and gas chromatography of the extracted steroids and their sylil and acetyl derivatives and by recrystallizations with authentic and acetylated unlabelled steroids. Several other steroids formed from acetate were tentatively identified. No androstenedione or testosterone were formed. That the Sertoli cell cultures were free of Leydig cells was established by the absence of histochemically detectable 3β-hydroxysteroid dehydrogenase activity and the inability of the cultures to oxidize the 3β-hydroxyl group of [¹⁴C]pregnenolone. This is the first direct evidence that Sertoli cells have the capacity to synthesize steroids $\text{de}$$\text{novo}$ from acetate.     

Kinetics of calcium efflux and exchange from Rana pipiens oocytes immediately following reinitiation of the first meiotic division: Comparison of various meiotic agonists and antagonists

The reinitiation of the meiotic divisions and the release of ⁴⁵Ca from the $\text{Rana}$$\text{pipiens}$ oocyte has been studied as a function of meiotic agonists and antagonists. Each of the meiotic agonists tested (progesterone, insulin, D-600, La³⁺) caused a decreased ⁴⁵Ca uptake and an increased efflux during the first 15 min after exposure. The effects of progesterone, D-600, and La³⁺ are not additive and progesterone will not release additional ⁴⁵Ca in oocytes pretreated with D-600 or La³⁺. Tetracaine inhibits both progesterone-induced release of ⁴⁵Ca and an early step in meiosis (nuclear membrane breakdown). [Tetracaine]_o required for 50% inhibition of nuclear breakdown decreases with decreasing [progesterone]_o suggesting competitive inhibition. The Ca, Mg-ionophore A23187 shows a similar competitive inhibition of progesterone-induced nuclear breakdown and stimulates a rapid release of ⁴⁵Ca within the first 1–3 minutes after exposure to the ionophore. Unlike progesterone, insulin, D-600, or La³⁺, the ionophore A23187 stimulates both uptake and efflux of ⁴⁵Ca by oocytes. These results suggest that both a reduced influx and a selective release of calcium from specific membrane sites is essential for steroid reinitiation of the meiotic divisions in $\text{R.}$$\text{pipiens}$ oocytes.     

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate stimulates lactate production in BALB/c 3T3 preadipose cells.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), which reversibly inhibits the adipose conversion of $\text{BALB}\text{c}$ 3T3 preadipose cells, increases lactate production by these cells. The stimulation of lactate production requires 4–7 days for optimal effect. Once TPA is removed from the cultures, the rate of lactate production falls to control levels. The concentration dependence for the TPA-mediated stimulation of lactate production is similar to that for its inhibitory effect on adipose conversion. Exogenous lactate in the absence of TPA also inhibits adipose conversion. These results suggest that the ability of TPA to interfere with the normal pattern of glucose metabolism may be important in the inhibitory effect of TPA on triglyceride accumulation in these cells.     

The herbicide glyphosate is a potent inhibitor of 5-enolpyruvylshikimic acid-3-phosphate synthase

The broadspectrum herbicide glyphosate (N-[phosphonomethyl]-glycine), which causes the accumulation of shikimic acid in plant tissues, inhibits the enzymatic conversion of shikimic acid to anthranilic acid in a cell-free extract of $\text{Aerobacter}\text{,}\text{aerogenes}$ 50% at 5 to 7 μM concentrations. Of the four enzymes involved in the transformation, only 5-enolpyruvylshikimic acid-3-phosphate synthase is inhibited by the herbicide.     

The effects of hypophysectomy and human chorionic gonadotropin administration on the in vitro metabolism of progesterone by the rat testis

Changes in the $\text{in}$$\text{vitro}$ capacity to convert progesterone to its metabolites were studied in testes of adult rats hypophysectomized for varying lengths of time. After 30 days of hypophysectomy rats were injected for periods of 10 and 20 days with 100 i.u. of HCG daily to observe what changes could be induced in the testicular conversion of progesterone. Hypophysectomy increased the formation of 20α-hydroxy-4-pregnen-3-one and decreased the formation of testosterone. In hypophysectomized animals injected with HCG there was an immediate decrease in the 20α-hydroxy-4-pregnen-3-one formation, but no appreciable accumulation of testosterone, as the animals demonstrated an immature pattern of testicular function. The results indicate that 20α-hydroxy-4-pregnen-3-one may act as a positive feedback agent to prolong and heighten gonadotropin discharge, and confirm the importance of metabolites of testosterone prior to adulthood.     

Nature of progesterone action on amino acid uptake by isolated full-grown oocyte of Xenopus laevis

l-leucine uptake into full-grown oocytes of Xenopus laevis is a saturable process which is Na⁺ dependent and presumably coupled to Na⁺ gradient. Our results indicate that progesterone (10^?6 M) blocks abruptly, around the germinal vesicle breakdown, the saturable transport of l-leucine. $\text{p-}\text{Chloromercuribenzoate}$ (10^?4 M) induces maturation and after a short lag of time strongly inhibits l-leucine uptake. Cycloheximide prevents progesterone-induced maturation and permeability changes.     

Monohydroxytamoxifen: an antioestrogen with high affinity for the chick oviduct oestrogen receptor

The monohydroxylated derivative of tamoxifen (a non-steroidal triaryl ethylene antioestrogen) shows an apparent affinity (Ki = 0.2 nM) for the chick oviduct oestrogen receptor which is higher than that of oestradiol itself, and ～ 10 times higher than that of tamoxifen. Administered $\text{in}\text{vivo}$ with oestradiol benzoate, it inhibited the increase of tissue growth, progesterone receptor content, ornithine decarboxylase activity (ODC), and ovalbumin and conalbumin synthesis, and also inhibited the oestradiol induced increase of ODC $\text{in}\text{vitro}$. It did not display any oestrogenic effect by itself. We conclude that antioestrogenic action may be exhibited by a molecule with higher affinity binding to the oestrogen receptor than oestradiol itself. Metabolic studies demonstrated that the antioestrogenic action of tamoxifen is not due to its prior conversion to monohydroxytamoxifen.