Showing papers on "Pregnenolone published in 1971"

On the Mechanism of Action of ACTH

Abstract: Publisher Summary This chapter focuses on the mechanism of action of adrenocorticotropic hormone (ACTH). The hormone stimulates steroidogenesis by a mechanism involving the synthesis of a protein with a rapid rate of turnover; the level of the protein then determines the rate of steroidogenesis. The experiments described in the chapter show that the inhibition of ACTH action by cycloheximide does not involve the inhibition of a step in the pathway prior to cholesterol but indicates that the antibiotic blocks steroidogenesis by preventing the further transformation of cholesterol in the pathway. The study presented in the chapter also demonstrates how the inhibition of protein synthesis blocked steroidogenesis by preventing the transformation of cholesterol to pregnenolone and the inhibitor had no effect on the other steps of the pathway. The injection of ACTH caused a marked decrease in adrenal cholesterol in association with its metabolism to the steroid hormones.

Secretion of free and sulfate-conjugated neutral steroids by the human testis. Effect of administration of human chorionic gonadotropin.

Abstract: Blood samples were obtained from the spermatic and peripheral veins of 8 males during an operation for inguinal hernia. Three of the subjects were treated with human chorionic gonadotropin (HCG) before collection of the samples. Neutral steroids in the fractions of free, mono- and disulfated compounds were identified and quantified, using gas-liquid chromatography and gas chromatography-mass spectrometry. In addition to testosterone, androstenedione and dehydroepiandrosterone, the following unconjugated neutral steroids were found to be secreted by the normal human testis: 5-androstene-3β,17α-diol, 5-androstene-3β,17β-diol, pregnenolone, 17α-hydroxypregnenolone and 17α-hydroxyprogesterone. In subjects treated with HCG, the concentrations of all these steroids in spermatic vein plasma were considerably higher than in untreated subjects. In addition, considerable amounts of monosulfated pregnenolone, dehydroepiandrosterone and 5-androstene-3β,17β-diol were found to be secreted by the testis in thes...

In vitro maturation and ovulation of oocytes of the catfish, Heteropneustes fossilis (Bloch): effects of mammalian hypophyseal hormones, catfish pituitary homogenate, steroid precursors and metabolites, and gonadal and adrenocortical steroids.

Abstract: Wolf and Quimby medium and its modified chemically-defined version were found to be most suitable among the eight media tested for shortterm culture of oocytes of the catfish, Heteropneustes fossilis. The modified Wolf and Quimby was employed to evaluate the relative effectiveness of several hypophyseal and steroid hormones, as well as steroid precursors and metabolites in inducing in vitro maturation and ovulation in the catfish oocytes. With the exception of ovine luteinizing hormone (LH) which gave only a marginal response even at high dose levels, other mammalian hypophvseal hormones (FSH, TSH, LtH, STH and ACTH) and catfish pituitary homogenate were incapable of inducing oocyte maturation. Of the steroid precursors tested, pregnenolone, progesterone and their 17 α-hydroxy derivatives were slightly effective in inducing oocyte maturation at high concentrations. Steroid metabolites (pregnanediol-20 β) were ineffective. The sex steroids (testosterone, estrone, estriol and estradiol) did not induce oocyte maturation, while dehydroepiandrosterone and androstenedione gave only a nominal response even at high doses. Corticosteroids, principally hydrocortisone (F) and desoxycorticosterone (DOC) as also their acetates, were the most potent maturation-inducing agents even at low concentrations. These results strongly suggest that inasmuch as LH and catfish pituitary homogenate per se do not induce oocyte maturation in vitro, the terminal hormones which act directly on the oocytes to induce maturation and ovulation are the corticosteroids principally, DOC and F. The significance of these findings in the hormonal control of ovulation in the catfish is discussed.

Biosynthesis of Pregnenolone

Abstract: Publisher Summary Pregnenolone is the C21 steroid closest in structure to cholesterol, which is the most abundant sterol in the Eutheria. C21 steroid is by far the major precursor of all the steroid hormones in the adrenal gland, ovary, and testis. Cholesterol sulfate converts to various steroidal sulfates in humans in vivo and to pregnenolone sulfate by bovine adrenocortical mitochondria in vitro. The chapter presents the most advanced scheme for the enzymatic transformation of cholesterol to pregnenolone. Cholesterol derivatives with only a hydroxyl at C-20 in the side chain appear not to have been isolated from natural sources. The premise that the hydroxylated derivatives of cholesterol served as better substrates than cholesterol itself was based on the more rapid transformation of the labeled precursors, which were not always studied at specified substrate concentrations. A better understanding of the mechanism of the conversion of cholesterol to pregnenolone awaits further investigation.

3β-Hydroxy steroid dehydrogenase activity in the mitochondria of rat adrenal homogenates

Abstract: The activity of 3β-hydroxy steroid dehydrogenase (EC 1.1.1.51) in the mitochondrial fraction of rat adrenal homogenates was approx. 31% of the total activity recovered after differential centrifugation and washing of the particulate fractions. Some 45% of the total activity was found in the microsomal fraction. The activity was assayed by a radioisotopic method devised in this laboratory for the purpose of studying small quantities of tissue and cell fractions. Satisfactory separation of the two fractions was demonstrated by electron microscopy of the pellets and by comparative recoveries of RNA, steroid 21-hydroxylase and cytochrome c oxidase in the various compartments. Analyses of the kinetics of the enzyme activity in the two fractions revealed no significant differences in apparent Km for pregnenolone, dehydroepiandrosterone or NAD+, but demonstrated a distinct difference in the Km for NADP+. pH optima and susceptibility to cyanoketone inhibition were similar in both fractions.

Studies on the structure and function of the mammalian testis III. In vitro steroidogenesis by the seminiferous tubules of rat testis

Abstract: The seminiferous tubules of rat testes were separated from the adjacent interstitial tissue by dissection and incubated with [7$\alpha $-$^{3}$H]pregnenolone and [4-$^{14}$C]progesterone in an oxygenrich, Krebs-Ringer bicarbonate medium at 33 degrees C for a maximum period of 6 h. Samples of the incubation medium were taken at various time intervals throughout the experiment to provide a kinetic study of the metabolic processes occurring, and the radioactive steroid products were identified and estimated. There was conversion of the labelled substrates to androgens and to a series of hydroxylated and/or reduced C$_{21}$ compounds, namely 20$\alpha $-hydroxypregn-4-en-3-one, 5$\alpha $-pregnane-3$\beta $,20$\alpha $-diol, 5$\alpha $-pregnane-3$\beta $-ol-20-one, 5$\alpha $-pregnane-3$\alpha $-ol-20-one, and 5$\alpha $-pregnane-3,20-dione. The preferred pathway of androgen synthesis was via $\Delta ^{4}$ intermediates. Thus the two series of compounds originated from the common precursor, progesterone, and then diverged along separate pathways. A comparison with parallel studies carried out previously on isolated interstitial tissue shows that there are definite differences in the steroidogenic activity of the two main components of the testis which cannot be accounted for by contamination. Hence it is concluded that the tubules possess a steroidogenic capacity distinct from that of the interstitium. It is suggested that the Sertoli cells are the most likely source of the androgens and perhaps also of the C$_{21}$ compounds. On this basis, and evidence which suggests that a significant proportion of the labelled metabolites were retained by the tubules (e.g. by selective binding), the possibility is considered that the capacity of the Sertoli cells to elaborate steroids in vitro may be at least equal to that of the Leydig cells.

In vivo production of steroids with central depressant actions by the ovary of the rat

Abstract: 1. In addition to progesterone and 20-dihydroprogesterone, the following unconjugated steroids were isolated from ovarian tissue and ovarian venous blood of mature, non-pregnant rats: pregnenolone, 3βOH-5α-pregnan-20-one, 3αOH-5α-pregnan-20-one, 20αOH-5α-pregnan-3-one and 5α-pregnane-3α, 20α-diol. 2. Their concentration in the ovarian tissue and their secretion rates into the ovarian venous blood was of the same order of magnitude as that of progesterone. 3. In the evening of the day of pro-oestrus significant increases in the ovarian contents of the following steroids were observed: progesterone, +140%; 3αOH-5α-pregnan-20-one, +320%; 20αOH-5α-pregnan-3-one, +195%; sum of pregnenolone and 3βOH-5α-pregnan-20-one, +275%. 4. At the same time the ovarian progeterone secretion rate was increased by 365%, that of 3αOH-5α-pregnan-20-one by 190%. 5. A possible physiological role of the ovarian allopregnane derivatives as central depressant agents is discussed.

Excretion of endogenous steroids and metabolites of (4-14C)pregnenolone in bile of female rats.

Abstract: Gas-liquid chromatography, gas chromatography-mass spectrometry and radio-gas chromatography were used to identify endogenous steroids and metabolites of [4-14C]pregnenolone in bile from female bile fistula rats. About 70% of the administered radioactivity was recovered in the bile during the first 24 h, and less than 1% during the following three days. 76% of the biliary radioactivity was extracted and was separated according to conjugation. The free steroid fraction contained 2%, the glucuronide fraction 22%, the monosulphate fraction 45%, and the disulphate fraction 28% of this radioactivity. The following steroids were identified by gas chromatography-mass spectrometry and were found to be labelled: glucuronide fraction: 3α-hydroxy-5α-androstan-17-one, 3α-hydroxy-5α,17β-pregnan-20-one and 3α,16α-dihydroxy-5α-pregnan-20-one; monosulphate fraction: 3α,7α-dihydroxy-5α-androstan-17-one and/or 3α,15α-dihydroxy-5α-androstan-17-one, 3α,11β-dihydroxy-5α-androstan-17-one and 3α,16α-dihydroxy-5α-pregnan-20-one; disulphate fraction: 3α,7α-dihydroxy-5α-androstan-17-one, 3α,11β-dihydroxy-5α-androstan-17-one, 5α-pregnane-3β,20β-diol and 3α,21-dihydroxy-5α-pregnan-20-one. The main part of radioactivity in the steroid monosulphate fraction was associated with pregnenolone that had a very high specific activity. The amount of steroids excreted during the first 24 h after bile duct cannulation was about 700 μg, when no correction for losses was made. The large quantity of steroid metabolites excreted in the bile confirms the importance of this excretory pathway also for endogenous steroids. The quantitatively most important steroids were found to be unlabelled corticosterone metabolites: 3α- and 3β,11β,21-trihydroxy-5α-pregnan-20-one and 3α,11β,15α,21-tetrahydroxy-5α-pregnan-20-one. Several other unlabelled steroids were identified. The heterogenous distribution of radioactivity among the metabolites is in contrast to the more uniform labelling previously obtained with intact rats. This shows that the metabolism of injected labelled steroids in bile fistula rats is not representative of the conditions in the intact animal, where enterohepatic circulation results in a continuous labelling of precursor steroid(s).

Steroidal androgen biosynthesis inhibitors.

Abstract: 2317beta-acylaminoandrost-4-en-3-ones and 3 previously known nonsteroids were synthesized and screened as inhibitors of 1720-lyase a step in androgen synthesis from progesterone or OH-progesterone. The screening involved measuring side chain cleavage (carbon-14-acetate release) from 21-carbon-14-17alpha-OH-progesterone by rat testis microsomes. The amide urea guanidino and carbamate derivatives were also tested by conversion of cholesterol to pregnenolone by a bovine corpora lutea acetone powder by conversion of corticosterone to 18-OH-corticosterone by crude adrenal mitochondria and by feeding to male rats to check effect on adrenal weight and testis testosterone level. More than 80% inhibition was achieved with androst-4-en-3-ones having the C-17beta carbamate formamido acetamido and ureido groups. These compounds did not inhibit OH-corticosterone synthesis. 6-alpha methylation inhibited the lyase 50-70%. 1 compound 17-beta-ureidoandrost-14-dien-3-one was fed to male rats for 6 weeks at 500 mg per kg; it reduced testis testosterone but not adrenal weight. Selective inhibition of androgen synthesis would be useful for treating benign prostate hypertrophy hirsutism acne and androgen dependent tumors.

Steroid metabolism by isolated rat seminiferous tubules in tissue culture.

Abstract: Cell monolayers were cultured from dissected rat seminiferous tubules in Eagle’s minimum essential medium for 7 days. The culture media were then replaced by fresh individual media containing pregnenolone-7α-3H, 17α-hydroxyprogesterone-7α-3H, dehydroepiandrosterone- 7α-3H and Δ5-androstenediol-7α-3H. After 48 hr incubation, the labeled steroid products were purified and identified by column, paper and thin layer chromatography, with derivative formation and crystallization to constant specific activity. The cell cultures showed minor conversion of pregnenolone to progesterone, 5α-pregnane-3,20-dione and dehydroepiandrosterone, and of 17α-hydroxyprogesterone to testosterone and androstenedione. By contrast, substantial conversions of dehydroepiandrosterone to testosterone and androstenedione, and Δ5-androstenediol to testosterone were observed. These findings show that cell cultures derived from seminiferous tubules possess the capacity for testosterone formation from proximate precursors and for reductive...

Gonadal Steroid Sulfates and Sulfatase. III. Correlation of Human Testicular Sulfatase, 3β-Hydroxysteroid Dehydrogenase-Isomerase, Histologic Structure and Serum Testosterone1

Abstract: To evaluate the role of steroid sulfates as precursors of testosterone in the human testis, the rate of cleavage of pregnenolone sulfate, dehydroepiandrosterone sulfate and androstenediol-3-sulfate was determined in the microsomal fraction of testes from 11 patients (27–80 yrs), three of whom had been on estrogen therapy prior to orchiectomy. The conversion of free Δ5-3β-hydroxysteroids to their Δ4-3-ketosteroid analogs, an obligatory reaction in testosterone biosynthesis, was determined separately in the same fractions with pregnenolone, dehydroepiandrosterone and androstenediol as substrates. Sections of each testis were examined histologically and Leydig cells quantitated. Serum testosterone levels were determined in 6 of the patients. Steroid sulfatase and 3β-hydroxysteroid dehydrogenase-isomerase activities were significantly reduced in the patients who had received estrogen therapy (Group I) when compared to those in non-treated patients (Group II). Mean “Leydig Cell Activity Index” (functi...

Identification and biosynthesis of steroid hormones in the ovary and fat bodies of female Triturus cristatus carnifex

Abstract: 1. 1. The steroid content of the ovaries and fat bodies of female Triturus cristatus carnifex have been analysed during the breeding season. 2. 2. Progesterone and androstenedione have been identified in the ovarian extract; the following steroids have been identified in the fat bodies: pregnenolone, progesterone, dehydroepiandrosterone, testosterone and estrone. 3. 3. Incubations of acetone powder carried out separately with ovarian and fat body tissue using cholesterol-4-C14 as the substrate yielded the following metabolites in the case of the ovary: progesterone, 17α-hydroxyprogesterone, androstenedione, testosterone; and in the case of the fat bodies: pregnenolone, 17α-hydroxypregnenolone, 17α-hydroxprogesterone, and testosterone. 4. 4. Incubations with either tissue using testosterone-4-C14 as the substrate yielded 11-ketotestosterone, 11β-hydroxyandrostenedione, androstenedione and esterone. 5. 5. These findings support the view that steroid hormones identified in the fat bodies could be produced in situ from cholesterol.

Studies on the mechanism of hrmonal stimulation of zinc uptake in human cell cultures: hormone-cell interactions and characteristics of zinc accumulation.

Abstract: Adrenal steroid hormones with glucocorticoid activity increase the uptake of Zn++ in HeLa cell cultures. On the basis of the level of Zn++ accumulation induced, steroid hormones can be classified into four groups: (a) optimal inducers (e.g., hydrocortisone and prednisolone); (b) suboptimal inducers (e.g., aldosterone and corticosterone); (c) anti-inducers (e.g., progesterone and 17 α-methyl testosterone) which competitively inhibit induction by optimal inducers; and (d) non-inducers (e.g., cortisone and pregnenolone) which neither induce nor inhibit the steroid-mediated increase in Zn++ uptake. The ability of an anti-inducer to block the effects of optimal inducers is not the result of inhibition of steroid uptake or an effect on general protein synthesis. Optimal inducers do not increase adenyl cyclase activity of HeLa cells nor can the hormone effects on Zn++ uptake be reproduced by 3'-5' cyclic AMP. The prednisolone-induced enhancement of Zn++ uptake is gradually lost over two or three days following removal of the hormone. Uptake of Zn++ by HeLa cells is not altered by a decrease of sodium concentration in the medium nor by changes in medium osmolarity. The uptake mechanism is not affected by subjecting intact cells to proteolytic enzymes; however, if cells are disrupted the hormone-mediated increase in Zn++ accumulation is lost. The Zn++ taken up by HeLa cells in the presence or absence of hormone is primarily cytoplasmic in localization and appears to be distributed in a multicompartmental system.

Steroid content of the equine ovary during the reproductive cycle

Abstract: It has been demonstrated that there may be some correlation between estrus and estradiol concentration in the Graafian follicles of mares. An investigation was undertaken to establish the differences in the steroid content of all the follicles of mares in estrus and to compare these differences with follicles at other stages of the cycle. Follicular fluid from individual follicles was collected from 9 mares in estrus and the concentration of the following steroids was determined by gas-liquid chromatography: progesterone 17alpha-hydroxyprogesterone androstenedione 19-norandrostenedione epitestosterone estrone and estradiol. The most vascular follicles which were probably those destined to ovulate tended to have the highest steroid concentrations. In these vascular follicles estradiol was the main steroid found with an average concentration of 151 mcg/100 ml. In 2 out of 3 samples of follicular fluid taken from the mares with recent ovulations estradiol was present in large quantities. The corpora lutea from these mares contained the following steroids: progesterone (average: 1973 mcg/100 g) 17alpha-hydroxyprogesterone (average: 65 mcg/100 g) 20alpha-dihydroprogesterone (average: 249 mcg/100 g) pregnenolone (average: 33mcg/100 g). Pregnenolone was not detected in any of the samples of follicular fluid examined. A pool of 8 midcycle corpora lutea contained the same 4 steroids as those in newly formed corpora lutea but in addition estradiol was detected in the order of 4 mcg/100 g.

Stimulation of pregnenolone synthesis by ACTH in rat adrenal sections.

Abstract: Pregnenolone accumulates in rat adrenal sections incubated in the presence of cyanoketone, and it is thus possible to directly study the net conversion of cholesterol to pregnenolone. ACTH and cyclic-AMP stimulate net pregnenolone synthesis to a degree which is sufficient to fully explain their effects on corticosterone production. Calcium and continued protein synthesis are directly required for the ACTHinduced stimulation of pregnenolone synthesis. Despite accumulation of pregnenolone within the adrenal tissue during incubation in media without added protein, there is no apparent inhibition of pregnenolone synthesis. The latter suggests that cholesterol side-chain cleavage is not significantly affected by the accumulated pregnenolone (Endocrinology 89: 958, 1971)

Studies on the Structure, Biosynthesis and Bacterial Metabolism of 15-Hydroxylated Steroids in the Female Rat

Abstract: Gas-liquid chromatography, gas chromatography-mass spectrometry and radio-gas chromatog-raphy have been used to obtain a tentative identification of eight 15-hydroxylated C21O4 steroids in faeces and urine from conventional female rats: 3α(and 3β),15α-dihydroxy-5α,14β-pregnane-11,20-dione, 3α(and 3β),11β,15α-trihydroxy-5α,14β-pregnan-20-one, 3α(and 3β),15α, 20β-trihy-droxy-5α,14β-pregnan-11-one and 5α,14β-pregnan-3α(and 3β),11β,15α,20β-tetrol. The assign-ment of stereochemistry is based on indirect evidence. The steroids were present in the free and monosulphate fractions and became labelled after administration of [4-14C]pregnenolone and [4-14C]corticosterone to the rats. Incubations in vitro with caecal contents from conventional rats showed that they were microbial metabolites of the 15α,21-hydroxylated C21O5 steroids previously shown to be present in faeces and urine from germfree rats.

Absorption and enterohepatic circulation of neutral steroids in the rat.

Abstract: The absorption of labelled pregnenolone, corticosterone, pregnenolone sulphate and deoxycorticosterone sulphate after intragastric, intraduodenal and intracaecal injection was studied in female bile fistula rats. Efficient absorption was observed in all cases. Evidence was obtained for the absorption of both free steroids and sulphates from the small intestine, free steroids being absorbed more rapidly. Partial hydrolysis of the sulphates, probably bacterial, occurred in the intestine. Radioactivity in the portal blood was distributed between the free, glucuronide, monosulphate and diconjugate fractions. Glucuronides were present in the bile but not in faeces and urine. The enterohepatic circulation of labelled pregnenolone and corticosterone was studied in a series of rats consisting of five animals connected by cannulae between the bile duct of one rat and the duodenum of the next rat. The average time for an enterohepatic circulation was calculated to be 3—4 h. The ratio between radioactivities eliminated in faeces and urine increased progressively and markedly with each enterohepatic cycle, as did the ratio between conjugated and free steroids. It is concluded that the steroids undergo extensive metabolism in each enterohepatic cycle and that labelled steroids injected into rats are unlikely to be fully mixed with the endogenous pools before being excreted.

Blood Progesterone and Pregnenolone Levels During Phenobarbital (PB) Block of PMS-induced Ovulation in Immature Rats1

Abstract: Phenobarbital (PB) block of ovulation in PMS-primed (day 30) immature rats is associated with altered blood levels of progesterone and pregnenolone. Progesterone decreased after administration of PB just prior to the critical period on day 32 and increased the next day, whereas pregnenolone was increased on the day of proestrus (day 32) and decreased the next day. Administration of progesterone before phenobarbital overcomes the PB block of ovulation and restores blood progesterone levels (during this PB block) to nearly the same levels and profile as that seen in the PMS-primed rats without PB. The pregnenolone levels were elevated during proestrus and decreased the next day in the progesterone-PB treated group. These data support and extend our previous in vitro findings that when PB is given just prior to the critical period it decreases the production of progesterone and increases the production of pregnenolone from ovarian tissue in PMSprimed rats. From these data and the present experiments it appea...