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.     

Early time sequence in pregnenolone metabolism to testosterone in homogenates of human and rat testis

The time sequence of the metabolism of [4-14C] pregnenolone to testosterone in homogenates of human and rat testis was studied with special emphasis on the chain of events in the early 15 min of incubation. The incubations were performed at 32 C in the presence of NAD and a NADPH-generating system. The various intermediate steroids were separated by means of HPLC using a silica aliphatic diol column. Correction for procedural losses was performed by dual labeling. The present study confirms earlier reported results which showed that in the rat metabolism of pregnenolone to testosterone proceeds via the delta 4 pathway. However, this discloses for the first time that the conversion of pregnenolone proceeds very fast: progesterone, 17 alpha-hydroxyprogesterone, and 17 alpha-hydroxypregnenolone as the only important delta 5 intermediate, peak and decline again to almost undetectable levels within the first 15 min of incubation. Androstenedione and testosterone start to accumulate from 1 min on under the conditions used. In contrast, in the human testis, homogenates metabolism of pregnenolone to testosterone proceeds comparatively slowly and almost exclusively via the delta 5 intermediates dehydroepiandrosterone and androstenediol. Testosterone makes its appearance only after about 8 min of incubation. The data illustrate the importance of short-term incubations in evaluating the metabolism of steroids.     

The sex pheromone precursor androsta-5,16-dien-3 beta-ol is a major early metabolite in in vitro pregnenolone metabolism in human testicular homogenates

In an earlier report we described the early time sequence of the in vitro metabolism of [4-14C]pregnenolone ([4-14C]P5) to testosterone in homogenates of human and rat testes and demonstrated the appearance of mainly delta 5 (humans)- and delta 4 (rats)-steroids within minutes after starting the incubation. In this study strong evidence is presented for the substantial synthesis from P5 of the sex pheromone precursor androsta-5,16-dien-3 beta-ol (ADL) in human, but not rat, testicular homogenates. The 16-unsaturated C19 steroid ADL appeared after 1 min of incubation, and within 5 min reached values (17-23% of total radioactivity added as [4-14C]P5) comparable to those of the major delta 5-steroids 17 alpha-hydroxypregnenolone and dehydroepiandrosterone. Thus, in humans, as in boars, the sex attractant precursor ADL is a major early testicular metabolite of P5.     

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.     

Plasma levels of ovarian steroids, including 17 alpha,21-dihydroxy-4-pregnene-3,20-dione and 3 alpha,17 alpha,21-trihydroxy-5 beta-pregnan-20-one, in female plaice (Pleuronectes platessa) induced to mature with human chorionic gonadotrophin

In one previous paper we reported on the identification of 11-deoxycortisol and 3 alpha,17 alpha,21-trihydroxy-5 beta-pregnan-20-one in the ovaries and plasma of mature female plaice and also described the development of radioimmunoassays for these two steroids. The present paper describes temporal changes in plasma levels of the free and conjugated forms of these and of some other steroids (17 alpha,20 beta-dihydroxy-4-pregnen-3-one, 17 alpha,20 beta,21-trihydroxy-4-pregnen-3-one, 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one, 17 alpha-hydroxy-4-pregnen-3-one, testosterone, and 17 beta-oestradiol) in female plaice injected with and without human chorionic gonadotrophin (HCG). Oocyte final maturation, but not ovulation, was induced by HCG injections. Levels of most of the steroids were also elevated by the HCG injections and were significantly higher than in control fish throughout the experiment (112 hr). The two most abundant steroids were 11-deoxycortisol and 3 alpha,17 alpha,21-trihydroxy-5 beta-pregnan-20-one (up to 600 ng ml-1). Only relatively small amounts of 17 alpha-hydroxy-4-pregnen-3,20-dione (less than 15 ng ml-1), 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, and 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one (less than 5 ng ml-1) were found. 17 alpha,20 beta,21-Trihydroxy-4-pregnen-3-one was not present. Testosterone and 17 beta-oestradiol levels rose briefly, in response to the first of the two HCG injections, and then fell significantly. The ratio of conjugated to free steroids (except for 17 alpha-hydroxy-4-pregnene-3,20-dione and 17 beta-oestradiol) was almost always greater than 1. In the HCG-injected fish, there was a significant negative correlation between the response of 17 beta-oestradiol levels and the response of 11-deoxycortisol and 3 alpha,17 alpha,21-trihydroxy-5 beta-pregnan-20-one levels. This further confirms that, as teleosts approach the time of full maturity, there is switch-over in the ovaries from predominantly C19 and C18 steroid production to predominantly C21 steroid production.     

Studies of the human testis. VII. Conversion of pregnenolone to progesterone.

Delta5-3beta-Hydroxysteroid dehydrogenase (EC.1.1.1.145) and steroid delta-isomerase (EC.5.3.3.1) were extracted from frozen human testicular tissue and co-precipitated by addition of ammonium sulfate. The activities of both enzymes were localized in the 0-40% (NH4)2SO4 fraction. The enzyme preparation catalyzed conversion of pregnenolone, 17alpha-hydroxypregnenolone, dehydroepiandrosterone, and androstenediol to the corresponding delta4-3-oxosteroid. Since isomerization appeared not to be the rate-limiting step of the overall reaction, measurement of activity of delta5-3beta-hydroxysteroid dehydrogenase was related to the amount of delta4-3-oxosteroid produced from the corresponding delta5-3beta-hydroxysteroid. Delta5-3beta-Hydroxysteroid dehydrogenase required NAD for maximal activity. The Michaelis constants (Km) for NAD were 50 muM, 33 muM and 14 muM, respectively for the dehydrogenation of pregnenolone, 17alpha-hydroxypregnenolone, androstenediol and dehydroepiandrosterone. Km values for each substrate were: pregnenolone 10 muM, 17alpha-hydroxypregnenolone and dehydroepiandrosterone 2.5 muM and androstenediol 3.0 muM. Human testicular delta5-3beta-hydroxysteroid dehydrogenase was inhibited by most of the steroids procued by the testis. The following steroids acted as competitive inhibitors with pregnenolone: 17alpha-hydroxypregenolone (Ki = 1.3 muM), androstenediol (Ki = 2.4 muM), dehydroepiandrosterone (Ki = 0.74 muM), 20alpha-dihydroprogesterone (Ki = 1.1 muM) estrone (Ki = 0.33 muM) and estradiol-17beta (Ki = 0.87 muM). 17alpha-Hydroxyprogesterone, testosterone and androstenedione showed mixed-type inhibition of the enzyme for pregnenolone. Progesterone and NADH were noncompetitive inhibitors of the enzyme for pregnenolone. Ki values, with respect to prenenolone, were 7.4 muM for progesterone and 150 muM for NADH. NADH, however, acted competitively with NAD and Ki value was 30 muM.     

Formation of 5alpha-reduced C19-steroids from progesterone in vitro by a pathway through 5alpha-reduced C21-steroids in ovaries of late prepubertal rats.

Ovarian homogenates from 10-150-day-old rats were incubated with [3H]progesterone and NADPH. Also, ovarian homogenates from 28-day-old rats were incubated for 5-180 min with either [14C]progesterone, [3H]5alpha-pregnane-3,20-dione or [14C]progesterone plus [3H]5alpha-pregnane-3,20-dione. Following incubation, radioactive metabolites were isolated, identified, and measured by column and paper chromatography, with derivative formation and recrystallizations to constant specific activity. Prepubertal ovaries (10, 20, and 28 days of age) converted 15-60% of progesterone to C21-17-hydroxysteroids and C19-steroids. At 40 and 150 days of age (postpubertal), the formation of these steroids decreased to less than 2%. At 10 and 150 days of age, the major C19-steroids formed from progesterone were androstenedione and testosterone. At 20 and 28 days of age, however, no accumulation of these C19-delta4-3ketosteroids was found (less than 0.1% of each), at which time the conversion of progesterone to 5alpha-reduced C19-steriods, such as androsterone and 5alpha-androstane-3alpha,17beta-diol, reached 30%. In ovaries of 28-day-old rats, the results from incubation studies for the detection of metabolic pathways indicated two biosynthetic pathways leading to 5alpha-reduced C19-steroids, one from progesterone via 5alpha-reduced C21 steroids, such as 3alpha-hydroxy-5alpha-pregnan-20-one and 3alpha,17alpha-dihydroxy-5alpha-pregnan-20-one, and a second via 17-hydroxyprogesterone, androstenedione, and testosterone. It seems that the active 5alpha-reduction of C19-delta4-3-ketosteroids and the formation of 5alpha-reduced C19-steroids by the pathway through 5alpha-reduced C21-steroids, are present in the ovaries of older prepubertal rats and may be the biological significance.     

Change of steroidogenic pathways in the ovary of a tropical catfish, Clarias macrocephalus, Gunther, after hCG treatment

Adult female catfish received an im injection of 454 IU hCG in 0.2 ml saline. Sixteen hours later, the ovarian tissue from the hCG-treated or control fish was aerobically incubated in vitro with 4-[14C]progesterone or 17 alpha-hydroxyprogesterone at 30 degrees for 60 min. When progesterone was employed as the substrate, significant production of androstenedione and testosterone was observed in the control group. However, after the hCG injection, a markedly higher amount of 20 beta-hydroxy-4-pregnen-3-one was produced. Furthermore, the androgen production was diminished, and the production of 5 beta-reduced C21 metabolites such as 5 beta-pregnane-3,20-dione and 3 alpha-hydroxy-5 beta-pregnan-20-one was also reduced in the hCG-treated group. From 17 alpha-hydroxyprogesterone as a substrate, considerable amounts of androstenedione and testosterone were obtained as the metabolites in the control group. However, after the hCG treatment, production of 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17 alpha, 20 beta-diOHprog) and its 5 beta-reduced metabolite was markedly stimulated, while the androgen production was reduced drastically. By evaluating the yield of each product, it was suggested that the tentatively calculated activity of 17 alpha-hydroxylase and C-17-C-20 lyase was diminished by the hCG treatment and that 20 beta-hydroxysteroid dehydrogenase was activated. It indicates that hCG changed the ovarian steroidogenic pathway from androgen production to formation of 17 alpha, 20 beta-diOHprog, an inducer of germinal vesicle breakdown.     

Plasma levels of ovarian steroids, including 17 alpha-20 alpha-dihydroxy-4-pregnen-3-one and 3 beta,17 alpha,20 alpha-trihydroxy-5 beta-pregnane, in female dabs (Limanda limanda)--marine flatfish--induced to mature and ovulate with human chorionic gonadotrophin

Human chorionic gonadotrophin (HCG) effectively stimulated oocyte final maturation and ovulation in female dabs (Limanda limanda) within 5 days of injection, and this was accompanied by significant changes in blood plasma steroid levels. The steroids which showed the greatest responses to the HCG injections were the ones previously found to be the major products of the ovaries in vitro: 17 alpha-20 alpha-dihydroxy-4-pregnen-3-one (17,20 alpha-P) and 3 beta,17 alpha,20 alpha-trihydroxy-5 beta-pregnane (3 beta,17,20 alpha-P-5 beta). 17,20 alpha-P responded more rapidly with peak levels after 32 hr of injection (115 ng ml-1), but 3 beta,17,20 alpha-P-5 beta reached higher levels ca. 12 hr later (320 ng ml-1). Levels of both steroids were not significantly different from initial values by the time of ovulation. 17 alpha,20 beta-Dihydroxy-4-pregnen-3-one, which is likely to be the oocyte maturation-inducing steroid (MIS) in the dab, showed a significant but very variable rise in levels (between 1 and 10 ng ml-1 in individual fish). 17 alpha-Hydroxy-4-pregnene-3,20-dione levels peaked at 6 ng ml-1 between 30 and 36 hr after HCG injection. Of the other C21 steroids identified in the ovaries of teleosts, 17 alpha,20 beta-21-trihydroxy-4-pregnen-3-one could not be detected, and 17 alpha,21-dihydroxy-4-pregnene-3,20-dione (11-deoxycortisol) showed nonsignificant changes compared to the saline-injected controls. HCG caused a decrease in estradiol-17 beta levels within 24 hr, but levels then rose again to a maximum of 8.2 ng ml-1 at ovulation time, possibly caused by the presence of vitellogenic oocytes in the ovaries. Changes in testosterone levels, however, were not significantly different between HCG- and saline-injected females. The role of HCG-responsive C21 steroids in the dab is discussed.     

Studies of the human testis. XVIII. Simultaneous measurement of nine intratesticular steroids: evidence for reduced mitochondrial function in testis of elderly men

To determine the basis for the decline in testosterone production by the aged testis, intratesticular unconjugated steroids, including testosterone, pregnenolone (3 beta-hydroxy-5-pregnen-20-one), 17 alpha-hydroxypregnenolone (3 beta,17 alpha-dihydroxy-5-pregnen-20-one), dehydroepiandrosterone (3 beta-hydroxy-5-androsten-17-one), androstenediol (5-androstene-3 beta,17 beta-diol), progesterone, 17 alpha-hydroxyprogesterone, androstenedione (4-androstene-3,17-dione), and 17 beta-estradiol, were measured by simultaneous RIAs in 32 previously untreated elderly men (aged 61-85 yr) undergoing orchiectomy as therapy for prostatic carcinoma and 20 young men (aged 25-35 yr) with oligospermia and varicocele. In vitro steroidogenesis using labeled pregnenolone as substrate was also investigated. Serum and intratesticular testosterone levels were lower (P less than 0.05) in aged patients [3.3 +/- 1.9 ng/ml and 0.86 +/- 0.53 microgram/g tissue (mean +/- SD)] than in young men (6.4 +/- 1.9 ng/ml and 1.7 +/- 1.1 microgram/g tissue), while circulating LH levels were higher (P less than 0.05) in elderly men (151 +/- 105 ng/ml) than in the young men (79 +/- 33 ng/ml), indicating that a primary pathological process affects the senescent testis, producing a decline in testosterone production. Study of bioconversion of [3H]pregnenolone to delta 4 steroids, 17 alpha-hydroxysteroids, and C19 steroids as well as analysis of the relative amounts of intratesticular steroids, as determined by RIA, revealed no apparent differences in the process of microsomal steroidogenesis in elderly compared to that in young men. The sum of the nine measured intratesticular steroid concentrations per g tissue wt was significantly lower (P less than 0.05) in aged patients (1.94 +/- 0.93 microgram/g tissue), than in young patients (3.68 +/- 1.90 micrograms/g tissue). The sum of the nine intratesticular steroids measured was positively correlated (P less than 0.01) with circulating LH levels in both patient groups, and the slope of this regression line was 14-fold greater for young men than for elderly men. Since the total concentration of the nine measured steroids reflects the pregnenolone supplied by the mitochondria within Leydig cells, it appears that the decline in Leydig cell function in aged men is attributable to a reduced supply of mitochondrial steroid precursors rather than to an impairment in microsomal steroidogenesis.     

Peripheral plasma concentrations of pregnenolone sulphate, pregnenolone, progesterone and 20 alpha-hydroxy-4-pregnen-3-one in ewes throughout the oestrous cycle

Pregnenolone sulphate, pregnenolone, progesterone and 20 alpha-hydroxy-4-pregnen-3-one concentrations in peripheral plasma of normal cyclic ewes were measured by radioimmunoassay. The concentrations of these steroids were correlated with that of progesterone. The concentrations of all the steroids measured in peripheral plasma varied in a cyclic manner and showed a significant (P less than 0.05) positive correlation with the concentration of progesterone. Peripheral plasma concentrations of these steroids in ovariectomized and ovariectomized, dexamethasone-treated ewes were also determined. The plasma concentration of progesterone in ovariectomized ewes was undetectable but the concentrations of pregnenolone sulphate, pregnenolone and 20 alpha-hydroxy-4-pregnen-3-one remained similar to those observed at oestrus. Administration of dexamethasone to ovariectomized ewes had no effect on pregnenolone sulphate or pregnenolone concentrations but 20 alpha-hydroxy-4-pregnen-3-one concentrations, which were already very low, decreased further. It is proposed that the ovary, probably the corpus luteum, secretes pregnenolone sulphate, pregnenolone and 20 alpha-hydroxy-4-pregnen-3-one; however, pregnenolone sulphate and 20 alpha-hydroxy-4-pregnen-3-one may also arise from the metabolism of circulating pregnenolone and progesterone.     

Identification of, and development of radioimmunoassays for 17 alpha, 21-dihydroxy-4-pregnene-3,20-dione and 3 alpha,17 alpha, 21-trihydroxy-5 beta-pregnan-20-one in the ovaries of mature plaice (Pleuronectes platessa)

Ovaries from a female plaice (Pleuronectes platessa) that had been injected with human chorionic gonadotrophin were incubated in vitro with 17 alpha-hydroxy[1,2,6,7-3H]progesterone. The major steroids produced by the ovaries were tentatively identified as 17 alpha,21-dihydroxy-4-pregnene-3,20di-one (11-deoxycortisol; 17,21-P), 17 alpha,21-dihydroxy-5 beta-pregnane-3,20-dione (3 alpha, 17,21-P-5 beta). A high proportion of these steroids was found in a conjugated form (sulphates or glucuronides). Radioimmunoassays were developed for 11-deoxycortisol and for 3 alpha,17,21-P-5 beta and were applied to fractions of mature male and female plaice plasmas and plaice ovarian incubates that had been separated on thin-layer chromatography. The presence of all three steroids, in vivo and in vitro, was confirmed. Particularly high amounts of conjugated 3 alpha,17,21-P-5 beta were found in the plasma of mature females (200-400 ng ml-1). The 3 alpha,17,21-P-5 beta radioimmunoassay also identified 3 alpha,17 alpha-dihydroxy-5 beta-pregnane-20-dione in all three fluids, despite the fact that this steroid was not among the radioactive incubation products of the ovary. These findings are compared with those from another flatfish, the dab (Limanda limanda), where the major gonadal steroids have been shown to be 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one and its 5 beta-pregnane (3-keto and 3 beta-hydroxyl) metabolites.     

Partial 17, 20-desmolase and 17 alpha-hydroxylase deficiencies in a 16-year-old boy

Thirteen plasma steroids as well as ACTH, LH and FSH were measured by specific RIAs under basal and dynamic conditions in a 16-year-old boy (normal external genitalia, 46, XY karyotype) who presented slowness and unachievement of pubertal development. On the delta 4-pathway: basal levels of testosterone and dihydrotestosterone were low- with a normal ratio-, delta 4-androstenedione and 11 beta-hydroxyandrostenedione were in the low normal range. Meanwhile, 17 alpha-hydroxyprogesterone and progesterone levels were markedly elevated. On the delta 5-pathway: dehydroepiandrosterone was extremely low while 17 alpha-hydroxypregnenolone and pregnenolone were almost normal; dehydroepiandrosterone sulfate was subnormal while pregnenolone sulfate was normal. Cortisol, aldosterone were normal while ACTH was moderately increased. Basal and responsive levels of LH and FSH were markedly increased. ACTH stimulation induced a subnormal rise of cortisol and 11 beta-hydroxyandrostenedione, a low or absent rise of dehydroepiandrosterone, 17 alpha-hydroxypregnenolone, androstenedione and 17 alpha-hydroxyprogesterone contrasting with a marked rise of pregnenolone and progesterone. After hCG stimulation, responses were low for testosterone, extremely high for 17 alpha-hydroxyprogesterone with a normalisation of the 17 alpha-hydroxyprogesterone/progesterone ratio. Fluoxymesterone dramatically reduced the pathologically high basal levels of progesterone and 17 alpha hydroxyprogesterone. Dexamethasone induced only a minute decrease in the delta 4-progestagens, a marked decrease in pregnenolone, with a more than 80% reduction of 17 alpha- hydroxypregnenolone, dehydroepiandrosterone, dehydroepiandrosterone sulfate and androstenedione. These data suggest a defect involving the cytochrome P450 common to both 17 alpha-hydroxylase and 17, 20-desmolase activities.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro metabolism of 4-pregnenes in ovaries of a freshwater teleost,the ayu (Plecoglossus altivelis): Production of 17α,20β-dihydroxy-4-pregnen-3-one and its 5β-reduced metabolites,and activation of 3β- and 20β-hydroxysteroid dehydrogenases by treatment with a fish gonadotropin

In vitro steroidogenesis in the mature ovaries of a freshwater teleost, the ayu (Plecoglossus altivelis), was examined. Cell-free homogenates of the ovaries of untreated fish or fish treated with a salmon gonadotropin (SG-G100) were aerobically incubated with ¹⁴C-labeled progesterone, 17α-hydroxyprogesterone, or 17α,20β-dihydroxy-4-pregnen-3-one, in the presence of NADPH. (1) Untreated fish: Progesterone was converted to 3α,17α-dihydroxy-5β-pregnan-20-one (the major product), 17α-hydroxyprogesterone, testosterone, 3α-hydroxy-5β-pregnan-20-one, 17α-hydroxy-5β-pregnane-3,20-dione, 17α,20β-dihydroxy-5β-pregnan-3-one, and 5β-pregnane-3α,17α,20β-triol. 17α-Hydroxyprogesterone was also transformed to 3α,17α-dihydroxy-5β-pregnan-20-one (the major product), in addition to the above-stated metabolites. 17α,20β-Dihydroxy-4-pregnen-3-one was metabolized into 17α,20β-dihydroxy-5β-pregnan-3-one and 5β-pregnane-3α,17α,20β-triol. (2) Fish treated with the gonadotropin: Besides the metabolites mentioned above, 17α,20β-dihydroxy-4-pregnen-3-one was confirmed as a metabolite of progesterone, and enhanced production of 17α,20β-dihydroxy-5β-pregnan-3-one and 5β-pregnane-3α,17α,20β-triol was observed. 17α-Hydroxyprogesterone was transformed to 17α,20β-dihydroxy-4-pregnen-3-one and 5β-pregnane-3β,17α,20β-triol, in addition to the metabolites obtained by the untreated fish, and formation of 17α,20β-dihydroxy-5β-pregnan-3-one and 5β-pregnane-3α,17α,20β-triol was increased. 17α,20β-Dihydroxy-4-pregnen-3-one was converted into 5β-pregnane-3β,17α,20β-triol besides 17α,20β-dihydroxy-5β-pregnan-3-one and 5β-pregnane-3α,17α,20β-triol. From these results it is concluded that, by the treatment of the fish with gonadotropin, ovarian 3β- and 20β-hydroxysteroid dehydrogenases were selectively activated.     

The characterization of polar corticosteroids in the urine of the macaque monkey (macaca fascicularis) and the baboon (papio hamadryas).

Computerised gas chromatography-mass spectrometry was employed in the identification of polar corticosteroid metabolites excreted in the urine from the macaque monkey (Macaca fascicularis) and the baboon (Papio hamadryas). The following steroids were identified in significant amounts in the urine from both species: 3alpha,17alpha,20alpha, 21-tetrahydroxy-5beta-pregnan-11-one; 3alpha,17alpha,20beta,21-tetrahydroxy-5beta-pregnan-11-one; 5beta-pregnane-3alpha,11beta,17alpha,20alpha,21-pentol; 5beta-pregnane-3alpha,11beta,17alpha,20beta-pentol; 5alpha-pregnane-3beta,11beta,17alpha,20beta,21-pentol. 11beta,17alpha,21-Trihydroxy-4-pregnene-3,20-dione (cortisol), 11beta,17alpha,20beta,21-tetrahydroxy-4-pregnen-3-one and 11beta,17alpha,20beta,21-tetrahydroxy-5xi-pregnan-3-one were identified in macaque monkey urine. Two steroids, 17alpha,20beta,21-trihydroxy-4-pregnane-3,11-dione and 17alpha,20alpha,21-trihydroxy-4-pregnene-3,11-dione were excreted as major C21 metabolites in the baboon but were not identified in the urine from the macaque monkey. 3beta-Hydroxy-5alpha-pregnane metabolites were identified in the urine from both species. All these steroids were excreted conjugated to glucuronic acid, evidenced by their recovery after hydrolysis with beta-glucuronidase enzyme. An efficient 20beta-reduction of corticosteroids in both species is apparent, and the excretion pattern of polar steroid metabolites in the two species was shown to be similar.     

Levels of 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one, 3 beta,17 alpha,20 alpha-trihydroxy-5 beta-pregnane, and other sex steroids, in blood plasma of male dab, Limanda limanda (marine flatfish) injected with human chorionic gonadotrophin

In a previous study, plasma sex steroid levels were measured in female dab (Limanda limanda) induced to ovulate by injections of human chorionic gonadotrophin (HCG). In the present study, a similar experiment was carried out on male dabs. In common with female dabs, 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one and 3 beta,17 alpha,20 alpha-trihydroxy-5 beta-pregnane showed the greatest response. Their plasma levels increased, respectively, from 6 +/- 1.6 and 13 +/- 6.2 ng/ml to ca. 62 ng/ml within 36 hr and then decreased. Levels of both steroids remained low in fish injected with saline. There was no statistically significant effect of HCG on plasma testosterone or 11-ketotestosterone concentrations. Initial levels of both hormones were between 10 and 20 ng/ml, and decreased simultaneously in both HCG- and saline-injected fish. Levels of 17 alpha-hydroxy-4-pregnen-3,20-dione, 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, 11-deoxycortisol, and 17 alpha,20 beta, 21-trihydroxy-4-pregnen-3-one were mostly below the detection limits of the assays (0.4 ng/ml). There was no statistically significant effect of HCG on either the total volume of milt collected or the proportion occupied by spermatozoa.     

Synthesis of 20 alpha-hydroxylated steroids by ovaries of the dab (Limanda limanda)

Dab (Limanda limanda) ovarian fragments were incubated in vitro with either [4,7-3H]pregnenolone or 17 alpha-hydroxy[1,2,6,7-3H]progesterone to investigate the pattern of steroidogenesis. A major enzyme found in the dab ovary was 20 alpha-hydroxysteroid dehydrogenase. Among the steroids that were tentatively identified in ovarian incubates were 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one (17,20 alpha-P). 17 alpha,20 alpha-dihydroxy-5 beta-pregnan-3-one, 3 beta, 17 alpha,20 alpha-trihydroxy-5 beta-pregnane (3 beta,17,20 alpha-P-5 beta), and 3 alpha,17 alpha,20 alpha-trihydroxy-5 beta-pregnane. The presence of these steroids in plasma of mature female and male dabs was studied by radioimmunoassay. The antiserum was raised against 17,20 alpha-P. The 17,20 alpha-[3H]P label was produced by incubating place milt with 17 alpha-hydroxy [3H]progesterone. The radioimmunoassay was shown to have a high cross-reaction with the 5 beta-reduced analogues of 17,20 alpha-P and was therefore used, in conjunction with thin-layer chromatography, to measure the steroids. High concentrations of both 17,20 alpha-P and 3 beta,17,20 alpha-P-5 beta found in female and male dab plasma. The possible role of these steroids is discussed.     

Lack of a direct effect of gonadotropin hormone-releasing hormone agonist on human testicular steroidogenesis

In an attempt to determine whether the chronic administration of GnRH agonist (GnRH-A) has a direct inhibitory effect on testicular steroidogenesis in the human, the testes of four men with disseminated prostatic cancer who were treated with GnRH-A daily for at least 1 yr were assayed for intratesticular pregnenolone (5-pregnen-3 beta-ol-20-one), progesterone, dehydroepiandrosterone, 17 alpha-hydroxypregnenolone (5-pregnen-3 beta 17 alpha-diol-20-one), 17 alpha-hydroxyprogesterone, androstenedione, and testosterone (T). In addition, testicular 17 alpha-hydroxylase, 17,20-desmolase, and 17 beta-hydroxysteroid dehydrogenase enzyme activities of the delta 4 pathway were measured. These intratesticular steroids and enzyme activities from four GnRH-A-treated patients were compared to those in five men (controls) who were orchiectomized as the primary treatment for their disseminated prostatic cancer and in three other men who were treated for 3-12 months with GnRH-A daily but received, in addition to the daily GnRH-A, 1000 IUhCG, im, every other day for 3 days immediately before their salvage orchiectomy, which was performed when their disease progressed. In the control group, the delta 5-steroids, particularly dehydroepiandrosterone and pregnenolone, represented the majority of the intratesticular steroids. Compared to control values, all intratesticular steroids except delta 4-P (for which there was no difference) were significantly lowered by treatment with GnRH-A. Intratesticular T was reduced by 98% from 328 +/- 139 (+/- SEM) ng/g testis in the control group to 8 +/- 3 in the GnRH-A-treated group (P less than 0.01). The additional treatment with hCG for 3 days in the GnRH-A-treated group reversed the inhibition of all steroids to either control or above control levels, with intratesticular T rising to 1144 +/- 273 ng/g testis. A similar trend was found for all three enzymatic activities, i.e., GnRH-A alone inhibited each of the enzymatic activities, whereas the addition of hCG reversed this inhibition by GnRH-A. These data indicate that the chronic administration of GnRH-A to elderly men results in inhibition in both the delta 4 and delta 5 pathways, with a subsequent decrease in the intratesticular T concentration. The ability of exogenous hCG to reverse both the reduction in delta 4 and delta 5 intratesticular steroid content and the intratesticular enzyme activities induced by GnRH-A treatment supports the concept that GnRH-A does not have a direct inhibitory effect on testicular T biosynthesis.     

Biosynthesis of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one in the ovaries of grey mullet (Mugil cephalus) during induced ovulation by carp pituitary homogenates and an LHRH analogue.

Three female mullets received a priming injection of carp pituitary homogenate followed by a resolving injection of an LHRH analogue 24 hr later. Ovarian biopsies were obtained just prior to the first injection (phase I), 24 hr after the first injection (i.e., immediately before the second injection, phase II), and 8 hr after the second injection (phase III). Two fish (Nos. 1 and 3) spawned approximately 12 hr after the second injection. Serum levels of testosterone increased to some extent during phase II in all of the fish. Testosterone levels decreased abruptly during phase III in both fish Nos. 1 and 3. In contrast the concentration of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHprog) was undetectable in phases I and II, and increased dramatically during phase III in the same fish. In fish No. 2, which did not spawn, neither the decrease of testosterone nor the increase of 17 alpha,20 beta-diOHprog was observed. Ovarian tissues of all the fish were pooled in each phase and incubated with 14C-labeled progesterone or 17 alpha-hydroxyprogesterone to investigate steroid metabolism. During phases I and II progesterone was converted to 17 alpha-hydroxyprogesterone, androstenedione, and testosterone. During phase III, production of these steroids decreased drastically, and in turn, synthesis of 20 beta-hydroxy-4-pregnen-3-one and 17 alpha,20 beta-diOHprog was induced. Using 17 alpha-hydroxyprogesterone as a substrate, androstenedione and testosterone were produced during phases I and II, whereas they decreased considerably during phase III. This was followed by the production of 17 alpha,20 beta-diOHprog as the major metabolite.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.