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)     

The effects of o,p'-DDD on human adrenal steroid synthesis

In the present study, the effects of o,p'-DDD on plasma levels of pregnenolone, 17 alpha-hydroxypregnenolone, progesterone, 17 alpha-hydroxyprogesterone, 11-deoxycorticosterone, deoxycortisol, corticosterone, cortisol, androstenedione and testosterone were studied in 6 patients with adrenal carcinoma (3 with Cushing's syndrome, 2 with adrenogenital syndrome, one without clinical manifestation) and 6 with Cushing's disease. Plasma levels of these steroids were decreased in all of the patients with adrenal carcinoma. The decrement of progesterone and 17 alpha-hydroxyprogesterone was greater than that of pregnenolone and 17 alpha-hydroxypregnenolone. These results indicate that o,p'-DDD inhibits both cholesterol cleavage enzyme and 3 beta-hydroxysteroid dehydrogenase coupled with delta 5 to 4 isomerase system. Plasma levels of pregnenolone and 17 alpha-hydroxypregnenolone showed a twofold increase on the 7th day after consecutive administrations of o,p'-DDD in patients with Cushing's disease. Plasma levels of cortisol were decreased to normal one month after continuous o,p'-DDD treatment. Urinary 17-OHCS and 17-KS have been decreased out of proportion to the decrease in plasma cortisol in the first week of o,p'-DDD treatment. Such a disparity suggests that o,p'-DDD might affect the extra-adrenal metabolism of cortisol. However, no evidence was found for the inhibition of hepatic C17-20lyase and glucuronyl transferase. Regression of pulmonary metastases was observed in one case with Cushing's syndrome due to adrenal carcinoma, suggesting that o,p'-DDD causes necrosis of the metastatic adrenal carcinoma. A remission of the disease was obtained in one patient with Cushing's disease after 6 months of continuous o,p'-DDD treatment. The usefulness of o,p'-DDD for the treatment of adrenal carcinoma with metastases and Cushing's disease was confirmed.     

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.     

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.     

Identification of nonecdysteroid steroids in hemolymph of both male and female Astacus leptodactylus (Crustacea) by gas chromatography-mass spectrometry

The O-pentafluorobenzyloxime (OPFB)-heptafluorobutyrylester (HFB) derivatives of hemolymph steroid extracts from both male and female Astacus leptodactylus were subjected to negative ion chemical ionization and capillary gas chromatography-mass spectrometry (NCI/GC-MS). Five nonecdysteroid steroids, namely pregnenolone, 17 alpha-hydroxypregnenolone, testosterone, cholesterol, and 6 beta-hydroxyprogesterone were identified. With selected ion monitoring (SIM), indications were found for the presence of four more steroids: androstenedione, 5 alpha-dihydrotestosterone, 11-ketotestosterone, and 11 beta-hydroxytestosterone. The presence of 6 beta-hydroxyprogesterone could only be demonstrated in the female hemolymph. With the technique used, dehydroepiandrosterone, progesterone, 17 alpha-hydroxyprogesterone, and estrogens could not be detected in male or female hemolymph extracts.     

In-vitro synthesis of androgen from pregnenolone in the testes of the goat (Capra hircus) and identification of 5-pregnene-3beta,17alpha,20alpha-triol as an intermediate in the metabolic pathway of pregnenolone

When [4(-14)C]pregnenolone was aerobically incubated in vitro in the presence of NAD+ and NADPH with cell-free homogenates of testicular tissue of adult domestic goats (Capra hircus), progesterone, 17alpha-hydroxypregnenolone, 17alpha-hydroxyprogesterone, 17alpha,20alpha-dihydroxy-4-pregnen-3-one, dehydroepiandrosterone, androstenedione and testosterone were identified as its known metabolites. Time-course studies on this metabolism showed that the production of 17alpha,20alpha-dihydroxy-4-pregnen-3-one and testosterone constantly increased up to the end of incubation, suggesting that these are both end-products of pregnenolone metabolism in this system. The other metabolites behaved as intermediates and were ultimately converted, in part, to testosterone by the testicular homogenates, indicating that testosterone was synthesized through both 4-ene and 5-ene-pathways. Furthermore, besides these metabolites, 5-pregnene-3beta,17alpha,20alpha-triol was also identified as an intermediary metabolite, formed from pregnenolone through 17alpha-hydroxypregnenolone in the presence of NADPH, and further convertible into 17alpha,20alpha-dihydroxy-4-pregnen-3-one by the microsomal fraction and into 17alpha-hydroxypregnenolone by the cytosol fraction in the presence of NAD+ and NADP+. It was not, however, significantly transformed into C19-steroids. Furthermore, 17alpha,20alpha-dihydroxy-4-pregnen-3-one, which was formed either from 5-pregnene-3beta,17alpha,20alpha-triol or from 17alpha-hydroxyprogesterone, remained almost unchanged without conversion to C19-steroids when incubated with the caprine testicular homogenates.     

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.     

HETEROGENEITY IN ADRENAL STEROIDOGENESIS IN NORMAL MEN AND WOMEN

Adrenal steroidogenesis has been studied in vivo in normal men and women. Serum levels of nine steroids on the biosynthetic pathway (the delta 5 3-beta-hydroxysteroids, pregnenolone (Pe), 17 alpha-hydroxypregnenolone (17Pe), dehydroepiandrosterone (DHEA), androstenediol (Adiol), and their delta 4 3-keto counterparts, progesterone (Po), 17 alpha-hydroxyprogesterone (17Po), androstenedione (Adione), and testosterone (T)) as well as cortisol were measured during adrenal suppression and stimulation. This study demonstrates a marked heterogeneity in adrenal steroid responses between different subjects in the normal population. Thus, in three subjects ACTH stimulation from a dexamethasone-suppressed state resulted in a far greater increment of 17Po than in the other nineteen normal subjects. These three individuals (designated Type 2 responders) may have a partial deficiency of 21-hydroxylase activity. In the remaining nineteen subjects (designated as Type 1 responders) the women had a greater increment of Adiol (P less than 0.05) and a lower increment of Po (P less than 0.01) than the men, suggesting that adrenal 3-beta-hydroxysteroid dehydrogenase/isomerase activity may be slightly lower in women than men.     

Mechanism of inhibition of human testicular steroidogenesis by oral ketoconazole

To determine the antisteroidogenic effect of ketoconazole (KTZ) in the human testis, we measured the plasma delta 5-pregnenolone, delta 5-17 alpha-hydroxypregnenolone, dehydroepiandrosterone (DHEA), progesterone, 17 alpha-hydroxyprogesterone, androstenedione (A), and testosterone (T) concentrations in three men with previously untreated metastatic prostate cancer at various time intervals for 24 h before and 48 h after the administration of 200 mg oral KTZ every 8 h. The adrenal glands of these three patients were suppressed (as measured by the plasma cortisol levels) by the administration of 1.0 mg dexamethasone daily for 7 days before and during the study. After six doses of KTZ, bilateral orchiectomy was performed, and the intratesticular concentration of the aforementioned seven steroids and the intratesticular activities of the 17 alpha-hydroxylase, 17,20-desmolase, and 17 beta-hydroxysteroid dehydrogenase enzymes in the delta 4-steroidogenic pathway were determined. These seven intratesticular steroids and three intratesticular enzyme activities were compared to those in five men with previously untreated prostate cancer who underwent orchiectomy as primary treatment for their disease. Plasma A, DHEA, and T all significantly decreased during KTZ therapy. There was no significant change in the other four steroids in the plasma. In the testis, delta 5-pregnenolone, delta 5-17 alpha-hydroxypregnenolone, and delta 4-17 alpha-hydroxyprogesterone were all significantly elevated, whereas intratesticular DHEA, A, and T were significantly decreased in the three KTZ-treated patients compared to levels in the five non-KTZ-treated patients. Measurement of the enzyme activities demonstrated a significant reduction in both 17 alpha-hydroxylase and 17,20-desmolase, but no change in 17 beta-hydroxysteroid dehydrogenase, in the KTZ-treated patients compared to the levels in the non-KTZ-treated patients. We conclude that oral KTZ decreases testicular T production by inhibiting the 17,20-desmolase and also the 17 alpha-hydroxylase steps in both the delta 4- and delta 5-T biosynthetic pathways.     

Mevinolin (lovastatin) inhibits androstenedione production by porcine ovarian theca cells at the level of the 17 alpha-hydroxylase:C-17,20-lyase complex

Mevinolin, putatively a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme-A reductase, was used to assess the contribution of de novo synthesized cholesterol to androgen production by ovarian thecal cells in vitro. Enzymatically dispersed thecal cells from 3- to 6-mm follicles of prepubertal gilts were incubated at 150,000 cells/ml with a maximally effective dose of LH (250 ng/ml) for 24 h. Mevinolin (3-50 microM) caused dose-dependent inhibition of androstenedione production. Addition of 25-hydroxycholesterol (0.025-25 microM) failed to restore androstenedione production to levels seen in the absence of mevinolin, suggesting an additional site of action of mevinolin beyond 3-hydroxy-3-methylglutaryl coenzyme reductase. The site of this inhibitory effect was determined by measuring steroid products formed in the presence of relevant steroid precursors. Mevinolin (12 microM) inhibited the production of 17 alpha-hydroxyprogesterone from progesterone and that of androstenedione from 17 alpha-hydroxyprogesterone, while 25-hydroxycholesterol to progesterone and pregnenolone to progesterone conversions were unimpaired. That mevinolin did not affect 3 beta-hydroxysteroid dehydrogenase:delta 5-delta 4-isomerase reactions was confirmed by demonstrating that conversions of pregnenolone, 17 alpha-hydroxypregnenolone, and dehydroepiandrosterone to progesterone, 17 alpha-hydroxyprogesterone, and androstenedione, respectively, were not affected by 12 microM mevinolin. These results indicate that mevinolin has an additional inhibitory action at the level of the 17 alpha-hydroxylase:C-17,20-lyase complex. The degree of inhibition of androstenedione production was not decreased with increased concentrations of progesterone or 17 alpha-hydroxyprogesterone substrate, suggesting that the inhibition was not competitive in nature. As the dose of mevinolin was increased up to 50 microM, progesterone accumulation was unaffected, but pregnenolone concentrations in medium greatly increased. While the mechanism of this effect is unclear, this finding suggests that preformed intracellular cholesterol, rather than that synthesized de novo, is supplying steroidogenic substrate in these cells.     

Secretion of testosterone and its delta4 precursor steroids into spermatic vein blood in men with varicocele-associated infertility

Insight into the mechanisms by which steroid hormones are released from the testes was sought by examining the concentrations of progesterone, 17alpha-hydroxyprogesterone, and androstenedione as well as testosterone in spermatic vein blood every 15 min for 4 h in men with varicocele-associated infertility. Coincident discrete secretory episodes of all four steroids were found, and spermatic vein concentrations of testosterone were highly positively correlated to the concentrations of progesterone (r = 0.79), 17alpha-hydroxyprogesterone (r = 0.81), and androstenedione (r = 0.82), respectively. The sum of the four measured steroids per mL plasma was calculated, and testosterone was found to account for 70%, 17alpha-hydroxyprogesterone for 24%, androstenedione for 5%, and progesterone for 1% of the total. In a previous study of the intratesticular steroids in a separate population of men with varicocele-associated infertility, the sum of these four steroids per g tissue was similarly calculated. Testosterone accounted for 70% of the four measured steroids, 17alpha-hydroxyprogesterone for 22%, androstenedione for 4%, and progesterone for 3% of the total. Thus, the relative concentrations of these four steroids are nearly identical in testicular tissue and spermatic vein plasma. From these data we hypothesize that steroids in the testicular interstitium are cosecreted into peripheral plasma in response to stimulation by LH and propose that the mechanism initiating this pulsatile mode of secretion oftestosterone and its precursor steroids may not be coupled to testosterone biosynthesis.     

Sexual differences of steroidogenic enzymes in embryonic gonads of the chicken (Gallus domesticus)

The left ovary and testis of 15-day-old embryos of the chicken were compared in the enzyme activities related to steroidogenesis. The activity of delta 5-3 beta-hydroxysteroid dehydrogenase coupled with delta 5-delta 4 isomerase in the ovary was similar to that of the testis. Activities of 17 alpha-hydroxylase and C-17-C-20 lyase in the ovary were 2.5 and 2.6 times those in the testis. From the CO-induced difference spectrum, the content of cytochrome P-450 in the ovarian microsomes was estimated as 27.6 pmol/mg protein. However, no detectable amount of cytochrome P-450 was observed in the testicular microsomal fraction. The substrate (progesterone)-induced difference spectrum was appreciable only in the ovarian microsomes. The activity of microsomal NADPH-cytochrome c reductase in the ovary was significantly higher than that in the testis. The activities of 17 beta-hydroxysteroid dehydrogenase in both gonads were similar to each other, when androstenedione was used as substrate. However, its activity in the ovary was 1.4 and 3.1 times that in the testis, when dehydroepiandrosterone and estrone were used as substrate, respectively. Aromatase activity in the ovary was over 100 times that in the testis, as assessed by release of [3H]water from [1-3H]testosterone. Appreciable amounts of radioactive estradiol-17 beta and estrone were formed from [4-14C]testosterone and [7-3H]androstenedione, respectively, only by the ovarian tissue. 5 beta-Reductase activity in the ovary was 1.4 times that in the testis.     

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.     

Effects of o,p'-DDD on pituitary-gonadal function in patients with Cushing's disease

O,p'-DDD has a cytotoxic action and inhibits the cholesterol side chain cleavage enzyme, 11 beta-hydroxylase, 3 beta-hydroxysteroid dehydrogenase coupled with delta 5 to 4 isomerase and 21-hydroxylase of the adrenal cells. However, the effects of o,p'-DDD on gonadal steroidogenesis are still unknown. In the present study, the effects of o,p'-DDD on Plasma cortisol, pregnenolone, 17 alpha-hydroxypregnenolone (17-OH-pregnenolone), progesterone, 17 alpha-hydroxyprogesterone (17-OH-progesterone), 11-deoxycorticosterone (DOC), corticosterone, dehydroepiandrosterone (DHEA), delta 4-androstenedione (androstenedione), estradiol, and LH and FSH were investigated in 3 patients with Cushing's disease before and after the administration of o,p'-DDD. The results are presented here. In Case 1 (18 yr old female) who had had secondary amenorrhea for 2 years, the plasma levels of cortisol, pregnenolone, 17-OH-pregnenolone, DHEA, androstenedione, testosterone, estradiol and corticosterone were elevated. The basal levels of plasma LH and FSH and the responses of both gonadotropins were lower than those of women with eumenorrhea. The plasma levels of progesterone, DHEA and testosterone decreased to normal 2 months after the beginning of the administration of o,p'-DDD. She restored menstrual cycles ranging from 40 to 50 days 3 months after the administration of o,p'-DDD, but with anovulatory bleeding. She showed a biphasic body temperature pattern with plasma progesterone and estradiol levels indicating corpus luteum formation 11 months after the start of the treatment, when plasma cortisol as well as progesterone and androgen were reduced to normal. The basal levels of FSH and LH and responses of these gonadotropins were slightly improved at that time. The plasma levels of cortisol, DHEA and androstenedione were high in Case 2 (38 yr old male) and Case 3 (45 yr old male), whereas plasma testosterone level was normal in Case 2 and low in Case 3. The plasma levels of these 3 steroids were normalized 28 days after the beginning of the o,p'-DDD administration. These results suggest that o,p'-DDD does not interfere with gonadal steroidogenesis in Cushing's disease.     

THE STEROID RESPONSE TO CONTROLLED ADRENAL STIMULATION IN CONGENITAL ADRENAL HYPERPLASIA

Adrenal steroidogenesis has been studied in vivo in eleven patients aged 13-68 years with 21-hydroxylase deficiency, in one patient with 11 beta-hydroxylase deficiency and in ten female control subjects. Serum levels of the delta 5 3 beta-hydroxysteroids, pregnenolone (Pe), 17 alpha-hydroxypregnenolone (17Pe), dehydroepiandrosterone (DHEA) and androstenediol (Adiol) and their delta 4 3-keto counterparts, progesterone (Po), 17 alpha-hydroxyprogesterone (17Po) androstenedione (Adione) and testosterone as well as of 11-deoxycortisol and cortisol were measured during acute adrenal suppression with dexamethasone followed by stimulation with synthetic 1-24 ACTH. In the seven patients with 21-hydroxylase deficiency who were on adequate glucocorticoid therapy, grossly exaggerated responses of 17Po and Po to ACTH were nevertheless preserved. In contrast, there was a grossly subnormal response of 17Pe, DHEA and Adiol to ACTH, and low basal levels of DHEA-sulphate. In the untreated patients the response of 17Pe and DHEA was normal. The Adione response was exaggerated in untreated and normal in treated cases. Similar findings obtained in the patient with 11 beta-hydroxylase deficiency who was studied after 6 weeks without replacement therapy. Our findings demonstrate that production of adrenal steroids that are associated with the adrenarche is not exaggerated in untreated CAH, and is grossly suppressed in treated cases. These findings are compatible with the hypothesis that intra-adrenal cortisol may initiate and/or maintain production of the delta 5 steroids by the zona reticularis that occurs in the human adrenarche.     

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.     

The 17, 20-lyase activity of cytochrome p450c17 from human fetal testis favors the delta5 steroidogenic pathway

Cytochrome P450c17 catalyzes both 17alpha-hydroxylation and 17,20-lyase conversion of 21-carbon steroids to 19-carbon precursors of sex steroids. P450c17 can mediate testosterone biosynthesis via the conversion of pregnenolone to dehydroepiandrosterone (the delta(5) pathway) or via conversion of progesterone to androstenedione (the delta(4) pathway). In many species, the 17, 20-lyase activity of P450c17 for one pathway dominates, reflecting the preferred steroidogenic pathway of that species. All studies of recombinant human P450c17 and of human adrenal microsomes have found high 17, 20-lyase activity only in the delta(5) pathway. Because the 17, 20-lyase activities in both the delta(4) and delta(5) pathways for testicular P450c17 have not been directly compared, however, it is not known if the delta(5) pathway dominates in the human testis. To resolve this issue, we assayed the conversion of 17alpha-hydroxypregnenolone to dehydroepiandrosterone (delta(5) 17, 20-lyase activity) and of 17alpha-hydroxyprogesterone to androstenedione (delta(4) 17, 20-lyase activity) by human fetal testicular microsomes. We obtained apparent Michaelis constant (K(m)) and maximum velocity (V(max)) values of 1.0 microM and 0.73 pmol.min(-1). microg(-1) for delta(5) 17, 20-lyase activity and of 3.5 microM and 0.23 pmol.min(-1). microg(-1) for delta(4) 17, 20-lyase activity. Catalytic efficiencies, expressed as the ratio V(max)/K(m), were 0.73 and 0.066 for the delta(5) and delta(4) reactions, respectively, indicating 11-fold higher preference for the delta(5) pathway. We conclude that the majority of testosterone biosynthesis in the human testis proceeds through the conversion of pregnenolone to dehydroepiandrosterone via the delta(5) pathway.     

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.     

The in vitro synthesis of final maturational steroids by ovaries of brook trout (Salvelinus fontinalis) and yellow perch (Perca flavescens)

The steroidogenic profile produced by ovaries of brook trout (Salvelinus fontinalis) and yellow perch (Perca flavescens) was examined using radioactive incorporation incubations. The radioactive metabolites produced were identified and were tested for their ability to induce germinal vesicle breakdown (GVBD) in oocytes in in vitro bioassays. Brook trout ovarian tissue converted [14C]progesterone to five metabolites that were effective in inducing GVBD in the bioassays: 5 beta-pregnanedione, 17 alpha-hydroxyprogesterone, deoxycorticosterone, and two more polar metabolites that were not identified. One of these two metabolites comigrated with 11-deoxycortisol and 17 alpha-hydroxy,20 beta-dihydroprogesterone (17 alpha, 20 beta prog) in thin layer chromatography. Androstenedione, testosterone, and estradiol-17 beta were also identified as metabolites of progesterone. Brook trout ovarian tissue did not extensively metabolize [14C]pregnenolone. Dehydroepiandrosterone and 17 alpha-hydroxypregnenolone were tentatively identified as metabolites of pregnenolone. Yellow perch ovarian tissue converted [14C]progesterone or [14C]pregnenolone to six metabolites that were effective in inducing GVBD in the bioassays: 5 alpha-pregnanedione, 17 alpha-hydroxyprogesterone, 20 alpha-dihydroprogesterone, 17 alpha-hydroxy, 20 alpha-dihydroprogesterone (tentatively identified), and two other unidentified metabolites. One of these two metabolites comigrated with 11-deoxycortisol and 17 alpha, 20 beta prog in thin layer chromatography. The only metabolite that induced ovulation of yellow perch oocytes in vitro was the metabolite comigrating with 11-deoxycortisol and 17 alpha, 20 beta prog. Androstenedione and testosterone were also identified as metabolites produced by yellow perch ovarian tissue. The results show that the ovaries of both species produce steroids that are active in the induction of oocyte final maturation.     

Steroidogenesis by cultured granulosa cells aspirated from human follicles using pregnenolone and androgens as precursors

Human granulosa cells from Graafian follicles aspirated 3-4 h before the expected time of ovulation were incubated with various steroid substrates, including pregnenolone, androstenedione, testosterone and dehydroepiandrosterone (DHA). Steroid production after 3 and 10 h of incubation was determined by radioimmunoassay. Progesterone and 17alpha-hydroxyprogesterone were the major products of granulosa cells in control short-term cultures with endogenous substrates. The addition of pregnenolone increased the synthesis of progesterone and 17alpha-hydroxyprogesterone compared with the controls, although the response varied considerably between paired short-term cultures. Little or no oestradiol-17beta was produced from endogenous precursors or short-term cultures to which pregnenolone had been added; one follicle, however, produced similar amounts of oestradiol-17beta in the control cultures and after incubation with pregnenolone. When granulosa cells were cultured with various amounts of androstenedione, DHA or testosterone, large amounts of oestradiol-17beta were produced, especially in short-term cultures in which larger amounts of substrate were added. Progesterone production continued and progesterone was synthesized more rapidly or in greater amounts in some short-term test cultures than in the controls. The results indicate that human granulosa cells are one source of oestradiol-17beta during the preovulatory phase. The data support the two-cell theory for oestradiol synthesis, for granulosa cells do not appear to undertake steroid conversion via the 5-unsaturated pathway, but aromatize androgens known to be produced by thecal cells. It is also suggested that either androgens or oestradiol-17beta stimulate progesterone production by granulosa cells, at least in vitro.