The effects of neonatal androgenization of male rats on testosterone metabolism by the hypothalamus-pituitary-gonadal axis

Male rats were androgenized on the third postnatal day by a single injection of 1 mg testosterone propionate. The in vitro metabolism of [4-14C]testosterone by pituitary and hypothalamus homogenates was investigated at the age of 90 days. The pituitary and hypothalamus homogenates from control and neonatally androgenized animals converted [4-14C]testosterone to the same metabolites, mainly 5 alpha-reduced derivatives; the quantitative yield of 5 alpha-reduced metabolites was much higher in the pituitary homogenates of androgenized rats. The hypothalamic homogenates showed no differences. In the androgenized rats a very significant increase of the plasma FSH levels was measured while the LH levels were also augmented. The plasma levels of testosterone were not different from the values in control rats, notwithstanding a 25% reduction in testes weight. The present experiments appear to indicate that the neonatal androgenization results in an accentuation of the sexual dimorphism which normally exists in the pituitary of adult rats for the 5 alpha-reductase activity.     

Influence of age on the formation of 5alpha-androstanediol and 7alpha-hydroxy-testosterone by incubated rat testes.

Testes from rats of different ages were indubated with or without tritiated testosterone. The exogenously-added or endogenously-produced testosterone is mainly metabolized to 7alpha-hydroxylated testosterone in adult animals, and to 5alpha-reduced metabolites (especially 5alpha-androstanediol) in immature animals.     

Formation of 5alpha-reduced C19 steroids from progesterone in vivo by 5alpha-reduced pathway in older prepubertal rat testis.

Either [3H] progesterone (0.5 or 5 nmol/5 muCi), 5alpha-[3H] pregnane-3,20-dione (5 nmol/5 muCi) or [14C] progesterone (6.6 nmol/0.2 muCi) plus 5alpha-[3H]-pregnane-3,20-dione (1 or 6.6 nmol/0.6 muCi), suspended in 0.05 ml of physiological saline solution, was injected into each testis of 32- and 90-day-old rats. Following injection, radioactive metabolites in testis and spermatic vein blood were extracted, isolated, measured and identified by column and paper chromatographies, with derivative formation and recrystallization to constant specific activity. In the blood and testis of older prepubertal rats, major 17-OH-C21 and C19 metabolites of progesterone were 5alpha-reduced steroids such as 3alpha, 17alpha-dihydroxy-5alpha-pregnan-20-one, 5alpha-androstane-3alpha,17beta-diol and androsterone. Following injection of [14C] progesterone plus 5alpha-[3H] pregnane-3,20-dione into 32-day-old rat testis, no significant augmentation of the isotope from progesterone was observed in 5alpha-reduced C19 steroids as compared with 5alpha-reduced 17-OH-C21 steroids, indicating that 5alpha-reduced C19 steroids were mainly formed from 5alpha-reduced 17-OH-C21 steroids in older prepubertal testis. In the blood and testis of adult rats, small amounts of 5alpha-reduced metabolites were shown to be produced from progesterone, while active 17alpha-hydroxylation of 5alpha-pregnane-3,20-dione followed by C17-C20-lyase reaction was demonstrated. These findings seem to indicate that formation of 5alpha-reduced C19 steroids from progesterone by the 5alpha-reduced pathway is a major pathway of androgen biosynthesis in older prepubertal rat testis in vivo.     

Studies on the kinetics of the interaction of 7 alpha-hydroxytestosterone with the steroid 5 alpha-reductase

Microsomal preparations from adult male rat testicular interstitial cells were incubated with tritiated testosterone. Added 7 alpha-hydroxytestosterone, (7 alpha,17 beta-dihydroxy-4-androsten-3-one), at levels which appear to exist in the adult testis, inhibited production of labelled 5 alpha-reduced steroids in a graded fashion. This interaction is not competitive and occurs only at high substrate levels, such as those found in steroid-producing organs. Relationships to pubertal changes in steroid metabolism are discussed.     

Influence of a prolonged tamoxifen administration on steroidogenesis by incubated rat testes

Tamoxifen was administered i.m. for 9 days to adult male rats in a daily dose of 100 micrograms or 1 mg. The treatment resulted in a significant reduction of the plasma levels of testosterone and LH, without modification of the plasma levels of FSH and of the testes weight. Upon incubation, the testes from the tamoxifen-treated rats produced less testosterone and 7 alpha-hydroxytestosterone, but metabolized [4-14C]testosterone in the same way as the control animals. Small doses of hCG (0.5 i.u. for 9 days) were unable to modify the tamoxifen effect on the testicular function, while tamoxifen significantly inhibited the increase of the plasma levels of testosterone induced by the administration of moderate doses of hCG (1.5 i.u. or 2.5 i.u. for 9 days) to hypophysectomized rats. Tamoxifen treatment, however, did not modify significantly the reactivity of the testes towards high doses of hCG (10 i.u.), administered either 2 h before sacrifice or for 9 days. It is concluded that a prolonged administration of tamoxifen in the rat has, besides an indirect effect resulting from a decrease of the LH levels, a direct inhibitory influence on the testicular testosterone formation, which can be reversed by high doses of hCG.     

Blood steroid profile and in vitro steroidogenesis by ovarian follicles and testis fragments of adult sea lamprey,Petromyzon marinus

The main purpose of the study was to identify the principal gonadal steroids synthesized by male and female sea lampreys, Petromyzon marinus. To achieve this, we used high performance liquid chromatography to separate the steroids in the serum of sexually mature animals, and to separate the steroids produced by gonadal tissue incubated in the presence of radiolabelled precursor steroids, as a means of identifying the major steroidogenic pathways. We were unable to detect evidence of the 'classical' steroids, such as 17beta-estradiol (E(2)) or testosterone (T) in the serum of either male or female lampreys. Instead, the principal chromatographic peaks contained very polar compounds that had elution times consistent with 15alpha-hydroxylated estrogens and androgens, and there were sex-specific differences in the chemical nature and the quantity of these compounds. Testis fragments or ovarian follicles co-incubated with tritium-labelled pregnenolone ([3H]P(5)), 17-hydroxyprogesterone ([3H]17OHP(4)), or androstenedione ([3H]A(4)), provided additional confirmation that the gonads synthesize a range of very polar steroids, and the metabolites found were consistent with the presence of a 15alpha-hydroxylated (15alphaOH) metabolic pathway common to testis and ovary. For ovarian tissue, the major 'end product' metabolites from all three precursors were 15alphaOH-estrogens, and for testis tissue 15alpha-hydroxyprogesterone (15alphaOHP(4)) and 15alpha-hydroxytestosterone (15alphaOHT) and small amounts of 15alphaOH estrogen. Small amounts of E(2) were also produced by both ovarian (all substrates) and testicular tissue (some substrates). Although it was assumed that the E(2) was synthesized via the aromatization of T, [3H]T was not found as an intermediate metabolite. The study suggests that the principal gonadal steroids in sea lamprey are 15alpha-OH compounds, and that only small amounts of E(2) or T are synthesized by the gonads at this stage of reproductive development. There was no direct evidence of progesterone (P(4)) synthesis from [3H]P(5), although the metabolites synthesized by both testis and ovary were indicative of a metabolic pathway that involved P(4) as an intermediate.     

Effects of chronic ethanol ingestion on steroid profiles in the rat testis

Testosterone, seven of its potential precursors, three of its metabolites and estradiol were analyzed in testes from rats given ethanol for 23 days in a nutritionally adequate liquid diet. The results were compared to those obtained with pair-fed control rats. The concentrations of pregnenolone, progesterone, 17-hydroxyprogesterone, androstenedione and testosterone were markedly lowered in four of the five rats given ethanol. The concentrations of the other 3 beta-hydroxy-delta 5 steroids and estradiol were unchanged, resulting in significantly increased ratios between 17-hydroxypregnenolone and 17-hydroxyprogesterone (P less than 0.025) and between androstenediol and testosterone (P less than 0.025) in the ethanol-treated rats. The results indicate that chronic ethanol administration reduces formation of testosterone by affecting a step prior to pregnenolone. There may also be an effect on the conversion of some 3 beta-hydroxy-delta 5 to the corresponding 3-oxo-delta 4 steroids. The levels of testosterone and three other steroids in testes of rats given the liquid diet were significantly lower than those in testes of animals fed a standard rat chow. This indicates a dietary influence on testicular steroid concentrations.     

The effects of various steroids on testosterone metabolism by the sexually mature rabbit epididymis

We examined the influences of steroids present in the epididymis on androgen metabolism by epididymal tissue and on the binding of androgen metabolites to the epididymal androgen receptor in castrated adult rabbit epididymides under in vitro conditions. The conversion of [3H]testosterone to [3H]17 beta-hydroxy-5 alpha-androstan-3-one (5 alpha-DHT) and to [3H]5 alpha-androstane-3 alpha (beta), 17 beta-diol was inhibited by unlabeled steroids in the following manner progesterone greater than testosterone greater than estradiol. Unlabeled 5 alpha-DHT did not inhibit [3H]testosterone metabolism indicating that product inhibition is not an important regulatory event. The antiandrogen cyproterone acetate did not inhibit the formation of 5 alpha-reduced metabolites of [3H]testosterone. All of the compounds used inhibited androgen binding to the classically defined cytoplasmic and nuclear androgen receptor.     

Inhibition of steroid 5 alpha-reductase and its effects on testosterone hydroxylation by rat liver microsomal cytochrome P-450

It has been shown previously that liver microsomal steroid 5 alpha-reductase activity increases with age in female but not male rats, which coincides with a female-specific, age-dependent decline in the cytochrome P-450-dependent oxidation of testosterone to 1 beta-, 2 alpha-, 2 beta-, 6 alpha-, 6 beta-, 7 alpha-, 15 beta-, 16 alpha-, 16 beta-, and 18-hydroxytestosterone and androstenedione. To determine whether the increase in steroid 5 alpha-reductase activity is responsible for the decrease in testosterone oxidation, we have examined the effects of the steroid 5 alpha-reductase inhibitor, 4-MA (17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one), on the pathways of testosterone oxidation catalyzed by rat liver microsomes. We have also determined which hydroxytestosterone metabolites are substrates for steroid 5 alpha-reductase. At concentrations of 0.1 to 10 microM, 4-MA completely inhibited steroid 5 alpha-reductase activity without inhibiting the pathways of testosterone oxidation catalyzed by liver microsomes from rats of different age and sex, and from rats induced with phenobarbital or pregnenolone-16 alpha-carbonitrile. 4-MA (10 microM) had little or no effect on the oxidation of testosterone catalyzed by liver microsomes from mature male rats (which have low steroid 5 alpha-reductase activity). In contrast, the hydroxylated testosterone metabolites formed by liver microsomes from mature female rats (which have high steroid 5 alpha-reductase activity) accumulated to a much greater extent in the presence of 4-MA. Evidence is presented that 4-MA increases the accumulation of hydroxytestosterones by two mechanisms. First, 4-MA inhibited the 5 alpha-reduction of those metabolites (such as 6 beta-hydroxytestosterone) that were found to be excellent substrates for steroid 5 alpha-reductase. In the absence of 4-MA, these metabolites eventually disappeared from incubations containing liver microsomes from mature female rats. Second, 4-MA inhibited the formation of 5 alpha-dihydrotestosterone, which otherwise competed with testosterone for oxidation by cytochrome P-450. This second mechanism explains why 4-MA increased the accumulation of metabolites (such as 7 alpha-hydroxytestosterone) that were found to be poor substrates for steroid 5 alpha-reductase. Despite its marked effect on the accumulation of hydroxylated testosterone metabolites, 4-MA had no effect on their initial rate of formation by liver microsomes from either male or female rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence for 15alpha- and 7alpha-hydroxylase activity in gonadal tissue of the early-life stages of sea lampreys, Petromyzon marinus

Gonads of premetamorphosing larval (PML), transforming (TL) and newly metamorphosed (juvenile) sea lampreys (JL) (Petromyzon marinus) were incubated in vitro with tritiated pregnenolone ([(3)H]P(5)), progesterone ([(3)H]P(4)), and androstenedione ([(3)H]A(4)) to identify the major products of steroidogenesis in early developmental stages. Reverse-phase high-performance liquid chromatography, using two mobile phase gradients, was used to separate the radioactive steroid metabolites. 7alpha-Hydroxylase activity was evident, based on the loss of radioactivity from [(3)H]P(5) labelled at position 7, appearing as tritiated water, and on the appearance of radiolabelled 7alpha-hydroxypregnenolone in the incubation medium. In addition, there was evidence of the synthesis of 15alpha-hydroxylated steroids from the three steroid precursors used. For the progestogen precursors, one of the major 15alpha -hydroxylated metabolites synthesized by both testis and ovarian tissue co-eluted with authentic 15alpha-hydroxyprogesterone, and for [(3)H]A(4), the product was predominantly [(3)H]15alpha-hydroxyandrostenedione. Additional polar steroids were produced, some of which co-eluted with authentic 15alpha-hydroxytestosterone and 15alpha-hydroxyestradiol, whereas others could not be correlated with the authentic 15alpha- or 15beta-hydroxylated steroids available. Ovarian tissues from PML and TL developmental stages synthesized several very non-polar compounds, some of which were present as unconjugated compounds, and others only in the conjugated fraction. These molecules had retention times consistent with pregnanes, and their presence in the incubation medium was therefore indicative of the presence of 5alpha-reductase. These metabolites were not present in the incubation medium from testis, or the JL ovary, suggesting that there is no expression of 5alpha-reductase activity in these tissues. Traces of 17beta-estradiol were found in the incubation medium from ovarian tissue incubated with P(5), but not following incubation with P(4) or A(4). Testosterone was not present in the incubation medium from either ovarian or testis fragments incubated with any of the substrates used.     

Biosynthesis and metabolism of testosterone by sertoli cell-enriched seminiferous tubules

Sertoli cell-enriched tubules isolated from rats which had been treated with 1,4-dimethyl sulfonyloxybutane were incubated with either [¹⁴C] progesterone or [¹⁴C] testosterone for 2 hours. Tubules of normal rats and fragments of Sertoli cell-enriched testes were incubated under the same conditions. Sertoli cell-enriched tubules converted progesterone to 20α-dihydroprogesterone, 17α-hydroxyprogesterone, androstenedione and testosterone. The major metabolite was 20α-dihydroprogesterone. The percentage conversion of progesterone into testosterone corresponded to a production of 10–20 ng testosterone. Sertoli cell-enriched tubules converted testosterone to dihydrotestosterone, androstenedione, 3α-androstanediol and 3β-androstanediol. Under our experimental conditions, dihydrotestosterone was the major 5α-reduced metabolite. Normal tubules converted progesterone and testosterone to the same metabolites as Sertoli cell-enriched tubules. Fragments of Sertoli cell-enriched testes were much more active than isolated tubules in metabolizing progesterone. They produced the same amounts of 5α-reduced metabolites of testosterone.     

Differences in steroid metabolism by testis, prostate and epididymis in the immature and adult possum (Trichosurus vulpecula)

Immature possums 126-195 days old and adults over 1 year old were used. Testicular homogenates from immature possums converted [3H]progesterone to nine different products, of which greater than 63% were 5 alpha-reduced androstane metabolites. The major product from adult testis was testosterone in yields greater than 60%. While metabolism of [3H]testosterone by the epididymis of immature possum was minimal, in adults 5 alpha-reduced products constituted greater than 80% of the yield. In contrast, prostatic tissue from adults converted less than 4% of [3H]testosterone to 5 alpha-reduced products, while the yields were greater than 80% from prostates of immature animals. The results showed that like in rodents, the testis of immature possum has a high 5 alpha-reductase and low 17 beta-hydroxysteroid dehydrogenase activity, which reverses in the adult state. The implications of the findings are discussed.     

Effect of neonatal estrogenization on testosterone metabolism in the prostate and in the epididymis of the rat

The present experiments were performed in order to analyze whether the administration of estrogens (single injection of 500 micrograms of estradiol benzoate s.c.) to neonatal male rats might modify the weight of the ventral prostate and the epididymis as well as the metabolism of testosterone in these two organs. The metabolism of testosterone was evaluated in vitro using 14C-radiolabelled testosterone as the substrate. The metabolites dihydrotestosterone (DHT), 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol), 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol), androstenedione, 5 alpha-androstane-3,17-dione (5-A-dione) and 3 alpha-hydroxy-5 alpha-androstane-17-one (androsterone) were quantified. After neonatal estrogen administration animals were killed on days 22 and 90 of age. The following changes were observed: (1) the body weight, the weight of the testes and of the ventral prostate were lower than in controls on both day 22 and 90; (2) the weight of the epididymides was higher than in controls on day 22 and lower on day 90; (3) in the ventral prostate the in vitro formation of DHT was lower and that of the diols was higher than in control tissue on day 22 of age; (4) the in vitro formation of alpha-reduced metabolites of the 17-keto series (5 alpha-A-dione + androsterone) was higher in ventral prostate of treated animals than in that of controls on day 22; (5) in treated animals, no formation of DHT in the caput epididymis was observed at day 22. On the contrary, at the same age the formation of androstenedione was higher than in controls; on day 90 of age the formation of DHT, androstenedione and the 5 alpha-reduced metabolites of the 17-keto series was identical in caput epididymis of the treated animals and of the controls, while the formation of the diols was higher in the treated than in the controls. The data indicate that neonatal estrogenization may induce important changes in testosterone metabolism in the prostates and in the epididymides of the rat.     

Modification of testicular steroid metabolism in the rat during gonadotrophin administration.

The administration of human chorionic gonadotrophins to adult rats stimulates the formation of testosterone, 7alpha-hydroxy-testosterone and 5alpha-androstanediol in incubated testes. When the gonadotrophins are injected for several days, the testosterone formation is maintained at a high level; however, the transformation to 7alpha-hydroxy-testosterone decreases progressively to subcontrol levels, while 5alpha-androstanediol is produced in greater amounts.     

The steroidogenic characteristics of primary testicular cell cultures from adult hypophysectomized rats: enhanced formation of C21 steroids

To characterize and clarify the time-related pattern of steroidogenesis in primary testicular cultures from adult hypophysectomized rats, we have determined the pattern of C19 and C21 steroids using novel enzymatic assay techniques that rely on highly specific bacterial hydroxysteroid dehydrogenases. Steroids contained in culture media were separated in a standardized high performance liquid chromatography system and the 17 beta-hydroxy- and 17-oxosteroids were quantified by a transydrogenase assay. The individual 3 alpha-, 3 beta-, 17 beta-, and 20 alpha-hydroxysteroids were in turn measured by enzymatic oxidation. Presumptive steroid identities were confirmed by enzymatic oxidation or reduction to products that were rechromatographed and identified by co-elution with standards. Although human chorionic gonadotropin stimulated an increase in the "adult" hormones, testosterone and 4-androstene-3, 17-dione, on both Days 1 and 11 of culture, the majority of the steroids found, even on Day 1, were 3 alpha-hydroxy-5 alpha-dihydrosteroids rather than delta 4-3-oxosteroids. A specific 5 alpha-reduced, C21 steroid: 5 alpha-pregnane-3 alpha, 20 alpha-diol, increased over time and became the most abundant gonodotropin-stimulated steroid (about 5-fold in excess of testosterone) by Day 11. In contrast, testosterone was the identifying steroid of nondispersed testes from both intact and hypophysectomized rats. Studies with tracer quantities of [3H]pregnenolone in culture confirmed the initial (Day 1) preponderance of 3 alpha-hydroxy-5 alpha-dihydrosteroids, as well as the accumulation with time of 20 alpha-hydroxysteroids. These findings suggest that contrary to expectation, cultured testicular cells from young adult hypophysectomized rats display a relatively atypical steroidogenic pattern. Although the cellular mechanisms underlying the time-dependent accumulation of C21 steroids remain uncertain, these patterns suggest either regressive changes in the original parent cells or the emergence of a population of latent cells. Although of limited utility as a model for examining adult testicular physiology, primary cultures of dispersed whole testes should prove useful in studies of culture-induced phenotypic regression and the attendant alteration at the level of gene expression.     

5alpha-reductase isoenzymes 1 and 2 in the rat testis during postnatal development

The pubertal initiation of spermatogenesis is reliant on androgens, and during this time, 5alpha-reduced androgens such as dihydrotestosterone (DHT) are the predominant androgens in the testis. Two 5alpha-reductase (5alphaR) isoenzymes (5alphaR1 and 5alphaR2) have been identified, which catalyze the conversion of testosterone to the more potent androgen DHT. The present study aimed to investigate the developmental pattern of 5alphaR isoenzymes and their relationship to the production of 5alpha-reduced androgens in the postnatal rat testis. Both 5alphaR1 and 5alphaR2 isoenzyme mRNAs were measured by real-time polymerase chain reaction, isoenzyme activity levels by specific assays, and testicular androgens by radioimmunoassay after high-performance liquid chromatographic separation. Both 5alphaR1 and 5alphaR2 mRNAs and activity levels were low in the 10-day-old (prepubertal) testis, peaked between Days 20 and 40 during puberty, and then declined to low levels at 60-160 days of age. The developmental pattern of both 5alphaR isoenzyme activity levels was mirrored by the testicular production of 5alpha-reduced metabolites. Although 5alphaR1 was greater than 5alphaR2 at all ages, it is likely, given the substrate preferences of the two, that both isoenzymes contribute to the pubertal peak of 5alpha-reduced androgen biosynthesis. The peak in 5alphaR isoenzymes and 5alpha-reduced metabolite production coincided with the first wave of spermatogenesis in the rat, suggesting a role for 5alpha-reduced metabolites in the initiation of spermatogenesis. This was explored by acute administration of a 5alphaR inhibitor (L685,273) to immature rats. The L685,273 markedly suppressed testicular 5alphaR activity during puberty by 75%-86%. However, a marked increase was observed in testicular testosterone levels (in the absence of changes in LH), and no decrease was observed in the absolute levels of 5alpha-reduced metabolites. Therefore, whether the formation of DHT in the presence of low testosterone levels in the pubertal testis is required for the initiation of spermatogenesis cannot be tested using 5alphaR inhibitors. We conclude that both 5alphaR1 and 5alphaR2 isoenzymes are involved in the peak of 5alpha-reduced androgen biosynthesis in the testis during the pubertal initiation of spermatogenesis.     

Androgen conversion in osteoarthritis and rheumatoid arthritis synoviocytes--androstenedione and testosterone inhibit estrogen formation and favor production of more potent 5alpha-reduced androgens

In synovial cells of patients with osteoarthritis (OA) and rheumatoid arthritis (RA), conversion products of major anti-inflammatory androgens are as yet unknown but may be proinflammatory. Therefore, therapy with androgens in RA could be a problem. This study was carried out in order to compare conversion products of androgens in RA and OA synoviocytes. In 26 OA and 24 RA patients, androgen conversion in synovial cells was investigated using radiolabeled substrates and analysis by thin-layer chromatography and HPLC. Aromatase expression was studied by immunohistochemistry. Dehydroepiandrosterone (DHEA) was converted into androstenediol, androstenedione (ASD), 16alphaOH-DHEA, 7alphaOH-DHEA, testosterone, estrone (E1), estradiol (E2), estriol (E3), and 16alphaOH-testosterone (similar in OA and RA). Surprisingly, levels of E2, E3, and 16alpha-hydroxylated steroids were as high as levels of testosterone. In RA and OA, 5alpha-dihydrotestosterone increased conversion of DHEA into testosterone but not into estrogens. The second androgen, ASD, was converted into 5alpha-dihydro-ASD, testosterone, and negligible amounts of E1, E2, E3, or 16alphaOH-testosterone. 5alpha-dihydro-ASD levels were higher in RA than OA. The third androgen, testosterone, was converted into ASD, 5alpha-dihydro-ASD, 5alpha-dihydrotestosterone, and negligible quantities of E1 and E2. 5alpha-dihydrotestosterone was higher in RA than OA. ASD and testosterone nearly completely blocked aromatization of androgens. In addition, density of aromatase-positive cells and concentration of released E2, E3, and free testosterone from superfused synovial tissue was similar in RA and OA but estrogens were markedly higher than free testosterone. In conclusion, ASD and testosterone might be favorable anti-inflammatory compounds because they decrease aromatization and increase anti-inflammatory 5alpha-reduced androgens. In contrast, DHEA did not block aromatization but yielded high levels of estrogens and proproliferative 16alpha-hydroxylated steroids. Androgens were differentially converted to pro- and anti-inflammatory steroid hormones via diverse pathways.     

Metabolism of testosterone sulfate in the rat: analysis of biliary metabolites.

Following intraperitoneal injection of a mixture of testosterone-7-3-H-17-sulfate and testosterone-4-14-C into male and female rats with bile fistulas, biliary metabolites were separated and purified by a combination of column chromatography, enzymic hydrolysis or solvolysis of the conjugate fractions and identification of the liberated aglycones. The injected steroids were extensively metabolized and excreted predominantly in the bile. The major portion of the 3H was excreted in the disulfate fraction in both sexes. Solvolysis of the disulfate revealed the sex-specific aglycone pattern: 5alpha-Androstane-3beta,17beta-diol was the major metabolite in the male rat, whereas 5alpha-androstane-3alpha,17beta-diol and polar steroids were found in the female. In marked contrast, testosterone was metabolized in a different way than testosterone sulfate. 14-C radioactivity was distributed in monoglucosiduronate, monosulfate, and diconjugate fractions. Analysis of the aglycones showed that polar steroids were the main metabolites in the male. In the female, testosterone was metabolized to polar steroids, androsterone, and 5alpha-androstane-3alpha,17beta-diol.     

Expression of cytochrome P450c17 and other steroid-converting enzymes in the rat kidney throughout the life-span

We have investigated the metabolism of [14C]-labelled progesterone (P4) and dehydroepiandrosterone (DHEA) by kidney tissues of newborn and 7-, 15-, 30-, 60- and 365-day-old rats of both sexes. The following enzymes were revealed at all ages by radiochemical identification of the corresponding products: 5alpha-reductase, cytochromes P450c17 and P450c21, 3beta-hydroxysteroid dehydrogenase (HSD)/delta5-delta4 isomerase, and 17beta- and 20alpha-HSDs, catalyzing reductive reactions. The major P4 metabolites were 5alpha-reduced C21 steroids, whose formation was almost completely suppressed by the 5alpha-reductase 4-azasteroid inhibitor, PNU 156765. Androstenedione and testosterone were also formed via 17alpha-hydroxyprogesterone, together with 11-deoxycorticosterone and 20alpha-dihydroprogesterone. DHEA was mainly converted to androst-5-ene-3beta,17beta-diol, with smaller amounts of the above androgens. Cytochrome P450c17 mRNA and protein were demonstrated by Northern blotting and Western blotting analyses, respectively. P450c17 mRNA, assessed by Northern blotting, protein and catalytic activity all peaked in the kidney samples at 15 days of life and declined thereafter. Cytochrome P450arom was below the level of detection of semi-quantitative RT-PCR. Since the rat kidney has been previously shown to contain cytochrome P450scc as well as androgen and estrogen receptors, it is suggested that it is capable of autonomous hormonal steroidogenesis and that renal steroids, or nephrosteroids, may act locally, in a paracrine or autocrine fashion.     

Conversion of 17 alpha-hydroxyprogesterone to 5 alpha, 3 alpha, and 20 alpha-reduced metabolites by female rat anterior pituitary and hypothalamus

The metabolism of 17 alpha-[3H]hydroxyprogesterone was examined in female rat anterior pituitary and hypothalamic tissues. After reverse isotopic dilution analysis and purification to constant specific activity, the following 5 alpha-, 3 alpha- and 20 alpha-reduced products were detected in both tissues: 17 alpha-hydroxy-5 alpha-pregnane-3,20-dione; 3 alpha,17 alpha-dihydroxy-5 alpha-pregnan-20-one; 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one and 5 alpha-pregnane-3 alpha,17 alpha,20 alpha-triol. While the metabolites formed were qualitatively the same, there were quantitative differences between the two tissues. The 3 alpha,5 alpha-reduced metabolite, 3 alpha,17 alpha-dihydroxy-5 alpha-pregnan-20-one, was the principal product in the anterior pituitary while the 5 alpha-reduced metabolite, 17 alpha-hydroxy-5 alpha-pregnane-3,20-dione, was produced in largest amount by the hypothalamus. With both tissues, the aforementioned four products plus starting substrate accounted for nearly all of the starting radioactivity. There was no evidence for the formation of C19 steroids (androgens) despite the presence of the 17 alpha-hydroxy group.