Pathway from progesterone to 5alpha-reduced c19 steroids not involving androstenedione and testosterone in immature mouse testes in vitro.

Testicular homogenates from mice of 23 and 70 days of age were incubated for 3-120 min with either 3-H-progesterone, 14-C-progesterone, 3-H-5alpha-pregnane-3,20-dione or 14-C-progesterone plus 3H-5alpha-pregnane-3,20-dione in the presence of NADPH. After incubation, radioactive products were purified and identified by column and paper chromatography, with derivative formation and recrystallization to constant specific activity. In immature mouse testes, the results indicate two biosynthetic pathways leading to C19 steroids from progesterone, one from progesterone via 17-hydroxyprogesterone and androstenedione to testosterone and a second via 5alpha-reduced C21 steroids to 5alpha-reduced C19 steroids such as androsterone and 5alpha-androstane-3alpha,17beta-diol. In adult mouse testes, very few 5alpha-reduced metabolites of all the delta-4-3-ketosteroids are shown to be produced from progesterone, while evident 17alpha-hydroxylation of 5alpha-pregnane-3,20-dione followed by C17-20 lyase reaction is demonstrated.     

Progesterone metabolism in vitro by rabbit testes at different stages of development.

Testicular homogenates from white rabbits of 0.4, 1, 3, 4, 5, 8, 18, and 24 months of age were incubated with [3H]progesterone and NADPH. At 12 days of age, major C21-17-OH-and C19- steroids formed from progesterone were 17alpha-hydroxyprogesterone and testosterone. However, at 4-24 months of age, 17alpha-hydroxyprogesterone, 3beta,17alpha-dihydroxy-5alpha-pregnan-20-one, and 5alpha-reduced C19-steroids such as 17beta-hydroxy-5alpha-androstan-3-one, 3beta-hydroxy-5alpha-androstan-17-one, and 5alpha-androstane-3beta,17beta-diol were the major products. The formation of significant quantities of 5alpha-reduced C19-steroids, which had been demonstrated previously only in prepubertal testes of rats and mice, was present in prepubertal as well as adult testes of rabbits. These findings clearly indicate that the metabolic patterns of progesterone in the adult rabbit differ from those in the adult rat and mouse. The major C19-steroids formed by testes are testosterone in the adult rat and mouse, but 5alpha-reduced C19-steroids in the adult rabbit.     

Relationship between the structures and steroidogenic functions of the testes of the urohaze-goby (Glossogobius olivaceus)

The testis of the brackishwater goby (Glossogobius olivaceus, the urohaze-goby in this text) consists of two main components, the glandular and the seminiferous tissue. After manual separation of the two tissues, in vitro steroidogenesis in each tissue was examined using testes from mature males in the breeding season. Cell-free homogenates (800g supernatant fluid) of each tissue were aerobically incubated with 14C-labeled pregnenolone, progesterone, 17 alpha-hydroxyprogesterone, androstenedione, dehydroepiandrosterone, testosterone, or 5 alpha-pregnane-3,20-dione in the presence of NAD+ or NADPH. (1) Glandular tissue: Pregnenolone and dehydroepiandrosterone were converted to progesterone and androstenedione, respectively, in the presence of NAD+. In the presence of NADPH, the following metabolism of steroids was established. Progesterone was transformed to 5 alpha-pregnane-3,20-dione (main product), 17 alpha-hydroxyprogesterone, 17 alpha-hydroxy-5 alpha-pregnane-3,20-dione, and androstenedione. 17 alpha-Hydroxyprogesterone was metabolized into 17 alpha-hydroxy-5 alpha-pregnane-3,20-dione (main product), 3 beta, 17 alpha-dihydroxy-5 alpha-pregnan-20-one, androstenedione, and 5 alpha-androstane-3,17-dione. From androstenedione, 5 alpha-androstane-3,17-dione (main product) and epiandrosterone were obtained. Testosterone was transformed to 5 alpha-dihydrotestosterone (main product), 5 alpha-androstane-3 beta, 17 beta-diol, epiandrosterone, and 5 alpha-androstane-3,17-dione. 5 alpha-Pregnane-3,20-dione was metabolized into 17 alpha-hydroxy-5 alpha-pregnane-3,20-dione, 5 alpha-androstane-3,17-dione, epiandrosterone, and 5 alpha-dihydrotestosterone. (2) Seminiferous tissue: Almost all of the above metabolites were obtained, but the yield was much smaller, especially for 5 alpha-reduced metabolites, compared with that for glandular tissue. From these results, it is concluded that steroidogenesis in the testis of G. olivaceus is characterized by the predominant activity of 5 alpha-reductase and 3 beta-hydroxysteroid dehydrogenase and that these are localized mainly in glandular tissue, together with delta 5-3 beta-hydroxysteroid dehydrogenase + delta 5-delta 4 isomerase, 17 alpha-hydroxylase, and C-17-C-20 lyase.     

Identification of two 5alpha-reduced pregnanes as major metabolites of progesterone in immature rat ovaries (1000 x g supernatant) in vitro.

Incubation of the 1000 x g supernatant obtained from 23-day-old rat ovarian homogenate with labeled progesterone resulted in the production of 3 major metabolites; 5alpha-androstane-3alpha,17beta-diol, and two 5alpha-reduced pregnanes that were identified as 3alpha-hydroxy-5alpha-pregnan-20-one and 3alpha,17alpha-dihydroxy-5alpha-pregnan-20-one. The 3alpha,17alpha-dihydroxy-5alpha-pregnan-20-one has not been hitherto isolated from mammalian ovaries. The steroids were identified by their mobilities on thin-layer and gas-liquid chromatography, by mass spectroscopy, derivative formation and by recrystallization to constant specific activity. In another experiment, incubation of the 1000 x g supernatant from 23-day-old rat ovaries with 3alpha-hydroxy-5alpha-pregnan-20-one as substrate resulted in the production of 5alpha-androstane-3alpha,17beta-diol. It is suggested that 5alpha-androstane-3alpha,17beta-diol is produced in immature rat ovaries by a pathway in which the identified 5alpha-reduced pregnanes serve as intermediates.     

Adrenal suppression with aminoglutethimide. III. Comparison of plasma delta 4- and delta 5-steroids in postmenopausal women treated for breast carcinoma.

A regimen or aminoglutethimide in combination with replacement glucocorticoid has been used to suppress adrenal steroidogenesis in postmenopausal women with metastatic breast carcinoma. During acute and chronic treatment with aminoglutethimide, the levels of the delta 4-steroids [progesterone (P), 17 alpha-hydroxyprogesterone (17-delta 4-P), and androstenedione (delta 4-A)] and the delta 5-steroids [dehydroepiandrosterone (DHEA), dehydroepiandrosterone-sulfate (DHEA-S), and 17 alpha-hydroxypregnenolone (17-delta 5-P)] were determine. In the total group of women, the plasma levels of P and delta 4-A increased 2- to 3-fold (P less than 0.05) while 17-delta 4-P rose 10-fold (P less than 0.01) from basal concentrations of 0.65 +/- 0.07 to 6.48 +/- 1.46 ng/ml during the initial 2 weeks of therapy with aminoglutethimide (AG) and dexamethasone. These three steroids then fell to basal levels during chronic treatment (P and 17-delta 4-P) or were suppressed (delta 4-A; P less than 0.001). In contrast, the levels of delta 5-steroids (17-delta 5-P, DHEA, and DHEA-S) were reduced 3- to 5-fold during the initial 2 weeks of therapy and remained suppressed throughout. The relative levels of certain delta 5- and delta 4-steroids pairs were then examined. The ratio of 17-delta 5-P to 17-delta 4-P decreased from baseline values of 2.15 +/- 0.35 to 0.38 +/- 0.21 ng/ml (P less .02) with the initiation of therapy and remained low thereafter. A similar pattern for the ratios between DHEA and delta 4-A, and DHEA-S and delta 4-A was observed. This may indicate that the regimen of AG treatment utilized may facilitate the activity of the 3 beta-ol-dehydrogenase, delta 5- to delta 4-isomerase, and accelerate the conversion of delta 5- to delta 4-steroids. The patterns of suppression of the plasma delta 4- and delta 5-steroids in oophorectomized and spontaneously postmenopausal patients with intact ovaries were analyzed separately. The plasma levels of progesterone were higher during the first 2 weeks of therapy in surgically castrate women than in spontaneously postmenopausal women (0.72 +/- 0.25 vs. 0.47 +/- 0.20 ng/ml). A similar pattern was observed for 17-delta 4-P, DHEA, and DHEA-S indicating that the adrenals might contribute to this increase. In contrast, during chronic treatment the levels of all steroids were lower in surgically castrate women than in those with intact ovaries. This suggested residual ovarian steroid during AG administration.     

Effects of multiple injections of LH on the secretion of pregnane compounds from polycystic ovaries of androgen-sterilized rats

Differences in the secretion of pregnane compounds by follicular polycystic ovaries of androgen-sterilized rats and by normal preovulatory ovaries of early pro-oestrous rats were compared. Some rats were injected i.v. with LH 30 min before bleeding, in order to stimulate the secretion of steroids. This injection of LH greatly increased the secretion of progesterone, 5 alpha-pregnane-3,20-dione and 3 alpha-hydroxy-5 alpha-pregnan-20-one by both types of ovaries. The response of the two progesterone metabolites in the polycystic ovaries was low, suggesting low 5 alpha-reductase activity. Because it is known that the preovulatory LH surge is absent in androgen-sterilized rats, a classical approach was taken to circumvent the probable deficit in cyclic release of LH by giving an i.v. injection of LH (25 micrograms) every 4 days for 16 days. Ovarian venous blood was collected 4 days after the last injection. The mean secretion of 5 alpha-pregnane-3,20-dione and 3 alpha-hydroxy-5 alpha-pregnan-20-one from the ovaries of such androgen-sterilized rats became much (P less than 0.01) higher than that of multiple saline-treated controls. These results suggest that low 5 alpha-reductase activity of polycystic ovaries in androgen-sterilized rats may be due to the absence of cyclic release of LH from the pituitary gland.     

Metabolism of (4-14C)progesterone and (7alpha-3H)pregnenolone by human ovaries perfused in vitro.

Nine human ovaries were perfused in vitro with [4-14C]progesterone and in addition one ovary with [7-3H]pregnenolone. From the perfusate unchanged progesterone and five different metabolites were isolated: 20alpha-dihydroprogesterone, 17alpha-hydroxyprogesterone, 16alpha-hydroxyprogesterone, 5alpha-pregnane-3,20-dione and 4-androstene-3,17-dione. In the ovarian tissue saturated pregnane derivatives were the main metabolites. When [3H]pregnenolone and [14C]progesterone were perfused simultaneously a stimulation of delta4-5-isomerase and 3beta-dehydrogenase activity by HCG was demonstrated.     

Periovulatory changes in steroid C17,20-lyase activity in ovaries of immature rats treated with pregnant mare serum gonadotropin

It has been established that the effect of LH on ovarian steroid formation results initially in a rise of overall steroid production, which 2-4 h later is followed by a decrease in the formation of C19-steroids. To determine the reason for this decrease, the course of 17 alpha-hydroxyprogesterone (17 alpha-OHP) metabolism in immature rat ovaries (10,000 X g supernatant) stimulated to ovulate with pregnant mare serum gonadotropin (PMSG) was investigated. Employing optimal conditions the C17,20-lyase was estimated from the formation of androstenedione from 17 alpha-OHP as substrate. The Michaelis-Menten constant Km was 61 +/- 4 microM and V varied according to the time after PMSG administration. The initial activity at 0 h was 340 pmol min-1 mg-1 protein. It decreased 48 h after PMSG to 160 pmol, followed by a slight rise at 54 h and followed by a 60% fall at 57 h, at the height of the LH-surge. The activity declined to undetectable levels at 60 and 64 h (8 h before ovulation) and remained undetectable after ovulation at 72 and 96 h after PMSG. The enzymes 5 alpha-reductase and 20 alpha-hydroxysteroid dehydrogenase were estimated from the formed 3 alpha, 17 alpha-dihydroxy-5 alpha-pregnan-20-one and 17 alpha, 20 alpha-dihydroxy-4-pregn-en-3-one, respectively. In the unstimulated ovary the 5 alpha-reductase was 405 pmol; it decreased to 110 pmol 48 h after PMSG and continued to decrease. The 20 alpha-hydroxysteroid dehydrogenase activity which was undetectable in untreated rats, rose to 330 pmol 60 h after PMSG, 8 h before ovulation. The changes in enzyme activities when tracer amounts of [3H] 17 alpha-OHP were used agreed with the pattern of kinetic studies. From these incubations eight metabolites were isolated, one of them, 5 alpha-pregnane-3 alpha,17 alpha, 20 alpha-trioi, has not been previously identified. It is concluded that the decrease in C17,20-lyase activity limit the formation of C19-steroids in preovulatory follicles shortly before ovulation.     

Steroid metabolism in normal mammary gland and in the dimethylbenzanthracene-induced mammary tumor of rats.

After incubation of [4-14C]progesterone with cell-free homogenates of 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced mammary tumor of rats, 20 alpha-hydroxy-4-pregnen-3-one, 5 alpha-pregnane-3,20-dione, 20 alpha-hydroxy-5 alpha-pregnan-3-one, 3 alpha-hydroxy-5 alpha-pregnan-20-one, and 5 alpha-pregnane-3 alpha, 20 alpha-diol were identified as the metabolites. In normal mammary tissue, however, 4-pregnene-3 alpha-diol was isolated in addition to 5 alpha-reduced, and 3 alpha- and 20 alpha-hydroxy metabolites. When radioactive testosterone was employed as a substrate, 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol were obtained as the metabolites of the mammary tumor. In the normal mammary gland, only 4-andorstene 3 alpha, 17 beta-diol was formed as its metabolite. Although the enzyme activities relevant to the metabolism varied among the tumor examined, the activity of 20 alpha-hydroxysteroid dehydrogenase in the mammary tumor was significantly lower than that in the normal mammary gland, whereas the activity of 5 alpha-reductase was higher in some of the mammary tumors than in the normal gland. The 5 alpha-reductase activity in the normal mammary gland was mostly localized in the crude microsomal fraction, whereas the same enzyme activity in the tumor was detected in all the organelle fractions. The activities of 20 alpha-hydroxysteroid dehydrogenase and NADPH-linked 3 alpha-hydroxysteroid dehydrogenase were found mainly in the cytosol fractions of the tumor and the normal tissue. The NADH-linked 3 alpha-hydroxysteroid dehydrogenase activity was detected only in the cytosol fraction of the normal mammary gland, but in the tumor studied, the activity of this enzyme was detected in all the subcellular fractions examined.     

Effects of multiple injections of luteinizing hormone on secretion of pregnane compounds from polycystic ovaries of rats exposed to constant light

Differences in the secretion of pregnane compounds by ovaries with cystic follicles of rats exposed to constant light (light-induced oestrous rats) and by ovaries with normal follicles of early pro-oestrous rats were studied. Some rats were injected iv with 2 micrograms of LH to stimulate the secretion of steroids 30 min before their blood was sampled. The injection greatly increased the secretion of progesterone by both kinds of ovaries. The secretion of 5 alpha-pregnane-3,20-dione and 3 alpha-hydroxy-5 alpha-pregnan-20-one also increased in normal rats, but not in the polycystic ovaries of light-induced oestrous rats, which suggested that the 5 alpha-reductase activity was low. The pre-ovulatory LH surge is absent in light-induced oestrous rats, so a classic approach was taken to circumvent the probable deficit in the cyclic release of LH; we gave multiple injections of 10 micrograms of LH. Five such injections were given at intervals of 4 days, and ovarian venous blood was collected 4 days after the last injection. Cystic follicles in the ovaries of rats disappeared when the injections of LH were given every 4 days. The production of 5 alpha-pregnane-3,20-dione and 3 alpha-hydroxy-5 alpha-pregnan-20-one from the ovaries of such rats was significantly higher (P less than 0.01) than in controls given multiple injections of saline. These results suggest that the low 5 alpha-reductase activity in polycystic ovaries of light-induced oestrous rats may be due to the absence of an LH surge from the pituitary gland.     

5 alpha-reduced androgens in the human fetal testis

The androgen content was measured in testes from 34 male and in ovaries from 30 female embryos that varied in age from less than 12 to approximately 20 weeks. The 5 alpha-reduced androgens dihydrotestosterone and 3 alpha-androstanediol were found in testes at a level of about a 30th of that of testosterone at all ages examined, whereas very little or no testosterone, androstenedione, or either of the 5 alpha-reduced androgens were detected in the ovaries. Whether dihydrotestosterone plays a role in the development of the testes is unknown.     

Seasonal changes in testicular steroidogenesis in the toad Bufo arenarum H

The biosynthesis of androgens in Bufo arenarum takes place through the 5-ene pathway that includes 5-androstane-3beta,17beta-diol as intermediate in testosterone biosynthesis. Besides testosterone and 5alpha-dihydrotestosterone, testes are able to synthesize 5alpha-pregnan-3,20-dione and several 3alpha- and 20alpha-reduced derivatives. Steroid biosynthesis changes during the breeding period (spring and early summer), turning from androgen to C21 steroid production. During the reproductive season, the production of progesterone, 5alpha-pregnan-3alpha,20alpha-diol, 3alpha-hydroxy-5alpha-pregnan-20-one, and 5alpha-pregnan-3,20-dione increases significantly. The function of most of these steroids in amphibians remains unknown. However, 5alpha-androstan-3alpha,17beta-diol and 3alpha-hydroxy-5alpha-pregnan-20-one were shown to be neuroactive in mammals, modulating sexual behavior. Thus, 5alpha/3alpha-reduced steroids could be involved in the regulation of the reproductive behavior in B. arenarum, a species with a dissociated reproductive pattern. Percentage contribution of each enzymes to the total metabolism reveals that neither 3beta-hydroxysteroid dehydrogenase/isomerase nor 5alpha-reductase change throughout the reproductive cycle. However, a strong reduction in 17-hydroxylase-C(17-20) lyase activity occurs in the reproductive season, suggesting that this enzyme could represent a key enzyme in the regulation of the seasonal change of steroidogenesis. Also, 3alpha-hydroxysteroid dehydrogenase and 20-hydroxysteroid dehydrogenase activities increase during the reproductive period, implying that steroid metabolism is clearly focused on C21-reduced steroids.     

In vitro biosynthesis of steroids from progesterone by the ovaries and pyloric ceca of the starfish Asterias rubens

In vitro biosynthesis of steroids from progesterone in ovaries and pyloric ceca of Asterias rubens has been investigated. The biosynthesis of 17α-hydroxyprogesterone, androstenedione, testosterone, 20α-dihydroprogesterone, 11-desoxycorticosterone, and 5α-pregnane-3,20-dione could be demonstrated to take place in the tissues of both organs by using [1,2-³H]progesterone as a precursor. The yields of intermediates of the Δ⁴-pathway and of 11-desoxycorticosterone are small, being higher in the ovaries than in the pyloric ceca. The yields of 20α-dihydroprogesterone are low, those of 5α-pregnane-3,20-dione are high. In both cases the yields in the pyloric ceca exceed those in the ovaries. The results indicate the presence of the following biosynthetic enzyme systems in ovaries and pyloric ceca of Asterias rubens: 17α-hydroxylase, C₁₇C₂₀-lyase, 17β-HSD, 20β-HSD, 21-hydroxylase, and 5α-reductase. The importance of these enzymes for the metabolism of progesterone, i.e., the biosynthesis of C₁₉-steroids, 20α-dihydroprogesterone, and corticosteroids, will be discussed.     

Progesterone and its reductive metabolism in steroidogenic tissues of the developing hen embryo

Progesterone contents of adrenals and ovaries and the--mainly reductive--metabolism of [3H]progesterone by these organs and liver were investigated in hen embryos between Days 13 and 21 (hatching). Progesterone contents are similar in adrenals and ovaries on Day 13 (approx 3.5 ng/mg) but descend in characteristic manners toward Day 17 and rise steeply, only in the ovaries, then descend in these organs toward hatching. [3H]Progesterone is converted by the adrenals toward 12 main metabolites, the main glucocorticoid being corticosterone (B), and the main reduced metabolite, 5 beta-pregnane-3,20 dione (5 beta-P). On day 13, 5 beta-P is five times as important as B, but both steroids evolve in a symmetric fashion, so that at hatching this proportion is reversed. In all tissues at all stages, except the liver on Day 13, the yields of 5 alpha-pregnane-3,20 dione (5 alpha-P) are one order of magnitude below those of 5 beta-P. Both diones exhibit maxima on Day 17, probably extending until Day 19. Concomitantly, [3H]progesterone disappearance is maximal on Day 17. Both ovaries differ in the shape of their 5 alpha-P/5 beta-P curves in that the left ovary exhibits for this curve a function ascending continuously toward hatching.     

Structure and steroidogenic enzymes of the seminal vesicles of the urohaze-goby (Glossogobius olivaceus)

The in vitro steroid metabolism in the seminal vesicles of the brackish water goby (urohaze-goby, Glossogobius olivaceus) was studied using males in the breeding season. The moderate activity of delta 5-3 beta-hydroxysteroid dehydrogenase was histochemically detected only in the epithelial cells of the organ, though these cells have the characteristics of secretory cells ultrastructurally. Cell-free homogenates (800 g supernatant fluid) of the whole tissue were aerobically incubated with 14C-labeled pregnenolone, progesterone, 17 alpha-hydroxyprogesterone, androstenedione, dehydroepiandrosterone, or testosterone in the presence of NAD+ or NADPH. Pregnenolone and dehydroepiandrosterone were converted to progesterone and androstenedione, respectively. Progesterone was transformed to 5 alpha-pregnane-3,20-dione (main product) and 17 alpha-hydroxyprogesterone. 17 alpha-Hydroxyprogesterone was metabolized into androstenedione (main product) and 17 alpha-hydroxy-5 alpha-pregnane-3,20-dione. From androstenedione, 5 alpha-androstane-3,17-dione (main product) and epiandrosterone were obtained. Testosterone was transformed to 5 alpha-dihydrotestosterone, 5 alpha-androstane-3 beta, 17 beta-diol, 5 alpha-androstane-3,17-dione, and androstenedione. These results indicate that the steroid metabolic patterns in the seminal vesicles of G. olivaceus are closely resembled to those in the testes.     

Stimulatory effect of prolactin on luteinizing hormone-induced testicular 5 alpha-reductase activity in hypophysectomized adult rats

Two pituitaries from 7-week-old female rats were grafted under the capsule of the left kidney of 50-day-old male rat to induce hyperprolactinemia. All of the pituitary-grafted and sham-operated rats were hypophysectomized at 56 days of age. The hypophysectomized rats in groups of 4 were given daily sc injections of saline or 9 micrograms NIADDK-ovine-(o)LH-23 for 4 and 5 days starting from days 58 and 70, respectively (short and long term hypophysectomized groups). The metabolism of [3H]progesterone or [14C]androstenedione by testicular homogenates, concentrations of testosterone and 5 alpha-androgens (androsterone plus 5 alpha-androstane-3 alpha, 17 beta-diol) in the serum and testes, and testicular LH receptors were estimated. Hypophysectomy caused significant decreases in testicular enzyme activities per gram of tissue, androgen production, and testicular LH receptors. In the testes of hypophysectomized rats, LH treatment significantly stimulated 5 alpha-reductase and 17-hydroxylase activities. Although pituitary grafts alone showed little or no effect on these testicular enzyme activities, hyperprolactinemia induced by the grafts markedly enhanced the LH-stimulated 5 alpha-reductase activity in both groups, especially in the long term hypophysectomized group. Therefore, androsterone and 5 alpha-androstane-3 alpha,17 beta-diol were shown to be the major C19-steroid products (immature type of testicular androgen production) in the LH- and PRL-stimulated testes of long term hypophysectomized adult rats. On the other hand, hyperprolactinemia was associated with a significant inhibition and a slight increase of the LH-stimulated 17-hydroxylase activities in the short and long term hypophysectomized groups, respectively. This difference can be attributed to both a PRL-induced increase in testicular LH receptors and a PRL-induced inhibition of 17-hydroxylase via a postreceptor mechanism(s). The present findings demonstrate for the first time that PRL directly stimulates LH-induced 5 alpha-reductase activity in the testes. It appears that PRL may play a role in the increased production of 5 alpha-C19-steroids and the parallel decrease of testosterone production in immature rat testes.     

Identification and quantitative determination of steroids in bovine corpus luteum during oestrous cycle and pregnancy.

Neutral steroids in bovine corpus luteum were isolated by liquid-gel chromatography on hydrophobic Sephadex, and were analyzed by computerized gas chromatography-mass spectrometry. The presence of progesterone and 20beta-hydroxy-4-pregnen-3-one was confirmed. In addition, 3beta-hydroxy-5-pregnen-20-one, 5-pregnene-3beta,20beta-diol, 3beta-hydroxy-5alpha-pregnan-20-one and 5alpha-pregnane-3beta,20beta-diol were fully identified, and 3-hydroxy-4-pregnen-20-one, 4 pregnene-3,20-diol, 22-hydroxycholesterol and 20,22-dihydroxycholesterol were partially characterized. Steroid sulphates were not detected. Quantification of the six fully identified steroids was based on peak areas in specific fragment ion current chromatograms constructed by the computer. During the 4th-19th day of the oestrous cycle the steroid concentrations varied as follows: progesterone 6.0-36.7 mug/g wet luteal weight, 20beta-hydroxy-4-pregnen-3-one 0.8-5.5 mug/g, 3beta-hydroxy-5-pregnen-20-one 1.0-7.1mug/g, 5-pregnene-3beta,20beta-diol smaller than 0.2-0.9 mug/g, 3beta-hydroxy-5alpha-pregnan-20-one 1.7-8.6 mug/g, and 5alpha-pregnane-3beta,20beta-diol smaller than 0.2-1.2 mug/g. The concentrations of progesterone and 3beta-hydroxy-5-pregnen-20-one seemed to vary in parallel and were low during days 11-17. During this period the concentrations of 5-pregnene-3beta,20beta-diol and 5alpha-pregnane-3beta,20beta-diol were highest as was the relative contribution of all three 20beta-hydroxysteroids to the total amount of steroids. The relative amount of 3beta-hydroxy-5alpha-pregnan-20-one seemed to be highest during days 4-6. The total steroid concentration in corpora lutea taken in early pregnancy (75-105 days) was 18-47 mug/g. In the period 75-90 days, progesterone constituted only 35-42% of the total steroids, 3beta-hydroxy-5alpha-pregnan-20-one as much as 23-40% and the total 20beta-hydroxysteroids 18-30%. The total steroid concentration in corpora lutea taken in midterm and late pregnancy was 21-77 mug/g. In this period progesterone was by far the predominant steroid and constituted about 80-90% of the total steroids in corpora lutea taken between days 150 and 240. Possible correlations between luteal growth, steroid oxidoreductases and steroid concentrations are discussed.     

Steroid 5alpha-reductase 1 promotes 5alpha-androstane-3alpha,17beta-diol synthesis in immature mouse testes by two pathways

5alpha-Androstane-3alpha,17beta-diol (androstanediol) is the predominant androgen in immature mouse testes, and studies were designed to investigate its pathway of synthesis, the steroid 5alpha-reductase isoenzyme involved in its formation, and whether testicular androstanediol is formed in embryonic mouse testes at the time of male phenotypic development. In 24-26-day-old immature testes, androstanediol is formed by two pathways; the predominant one involves testosterone --> dihydrotestosterone --> androstanediol, and a second utilizes the pathway progesterone --> 5alpha-dihydroprogesterone --> 5alpha-pregnane-3alpha-ol-20-one --> 5alpha-pregnane-3alpha,17alpha-diol-20-one --> androsterone --> androstanediol. Formation of androstanediol was normal in testes from mice deficient in steroid 5alpha-reductase 2 but absent in testes from mice deficient in steroid 5alpha-reductase 1, indicating that isoenzyme 2 is not expressed in day 24-26 testes. The fact that androstenedione and testosterone were the only androgens identified after incubation of day 16 and 17 embryonic testes with [3H]progesterone implies that androstanediol formation in the testis plays no role in male phenotypic differentiation in the mouse.     

Uptake and conversion of progesterone and testosterone to 5alpha-reduced products by enriched gonadotropic and chromophobic rat anterior pituitary cell fractions.

Monodispersed cells from anterior pituitaries of male rats were prepared by Pronasedissociation and incubated with [3H]progesterone or [3H]testosterone. The cells were then separated into enriched fractions consisting of gonadotropic, somatotropic or chromophobic cells by velocity sedimentation at unit gravity for 4 h. The uptake of [3H]progesterone and [3H]testosterone by the gonadotropic enriched cell fraction was 1.8 to 3.2 times greater than the somatotropic and chromophobic enriched cell fractions. The gonadotropic and chromophobic enriched cell fractions metabolized [3H]progesterone and [3H]testosterone appreciably. The principal metabolites were identified and quantitated by reverse isotopic dilution. After incubation with [3H]progesterone, the principal metabolite was [3H]5alpha-dihydroprogesterone which ranged from 11.5% for the gonadotropic cells to 7.6% for the chromophobic cells. Smaller amounts of 3alpha-hydroxy-5alpha-pregnan-20-one (3.7 to 4.8%) and 20alpha-dihydroprogesterone (2.1 to 4.3%) were also identified. After incubation with [3H]testosterone, the principal metabolite was 5alpha-dihydrotestosterone which ranged from 12.6% for the gonadotropic cells to 10.3% for the chromophobic cells. Smaller amounts of 5alpha-androstane-3alpha, 17beta-diol (4.1 to 5.5%) and androstenedione (1.8 to 3.0%) were identified. After incubation with [3H]progesterone or [3H]testosterone the same metabolites were also identified in the somatotropic cell fraction but were thought to be present because of contamination with gonadotropic cells. Dissociated pituitary cells from orchidectomized rats had a 2-fold increase in the uptake of [3H]testosterone and a greater metabolism of [3H]testosterone to 5alpha-dihydrotestosterone as compared to pituitary cells from intact rats (12.6 vs 25.7%).     

Ovarian steroidogenesis in vitro during the first month posthatching in the domestic chick: gonadotropin responsiveness and [3H]progesterone metabolism

Ovarian steroidogenesis was examined in domestic chicks during the first month posthatching. In vitro production of androstenedione during a 4-hr incubation was enhanced in a dose-dependent manner by 0.1-100 ng/ml of chicken LH (cLH). The greatest response was observed in ovaries from Day 1 chicks (eightfold), but cLH was also effective in Day 7, 14, 21, and 28 ovaries (three- to sixfold increase). Estrogen production in response to cLH was increased significantly only in Day 1 and 7 ovaries. A similar trend in androstenedione and estrogen production was seen in response to 8-bromo cAMP, oLH, and oFSH. In an additional experiment, in vitro metabolism of [3H]progesterone by ovaries from Day 1, 7, 14, and 21 chicks was examined during a 4-hr incubation. The major metabolite, comprising 18-19% of the radioactivity, coeluted with 5 beta-pregnan-3,20-dione. This is the first report of gonadotropin-stimulated steroidogenesis and progesterone metabolism in the chick during the first month post-hatching. The results show that the responsiveness to gonadotropins decreases during this period and the profile of [3H]progesterone metabolism does not resemble that seen in adult granulosa and theca cells.