Opposite effect of human chorionic gonadotropin on placental and ovarian synthesis of androstenedione

In the present studies, we have investigated the role of human chorionic gonadotropin on the biosynthesis of androgens by placentas and corpora lutea of the pregnant rat. We first sought to compare the effect of hCG on placental and ovarian secretion of androstenedione in vivo. For this purpose either 1.5 IU hCG or vehicle was administered to pregnant rats twice on days 12 and 13 and once on the morning of day 14. Blood was obtained from either the ovarian or the uterine vein. After hCG administration, levels of androstenedione secreted in the ovarian vein increased dramatically, whereas those in the uterine vein declined significantly. To establish that changes in androgen levels in the uterine and ovarian veins are due to changes in biosynthetic activity and also to compare the action of hCG on placentas and corpora lutea, tissues were dissected out from rats treated with 0, 1.5, or 9 IU hCG twice daily and incubated in vitro. hCG administration increased the capacity of luteal cells to synthesize androstenedione de novo by approximately 100% and concomitantly decreased placental secretion of androstenedione by approximately 75%. Addition of high density lipoprotein to the medium enhanced both basal and hCG-stimulated androstenedione production by luteal tissues but had no effect on either basal or hCG-inhibited androstenedione biosynthesis by the placenta. To determine which step in the placental biosynthesis of androstenedione is inhibited by increased levels of LH, we determined the effect of hCG administration, on cholesterol biosynthesis and storage, synthesis of progesterone substrate, and the activities of 17 alpha-hydroxylase/17,20-lyase. hCG did not affect the activities of the rate limiting cholesterogenic enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase, placental content of cholesterol and cholesteryl ester, or the placental production of progesterone. However, hCG did cause a substantial decrease in the activity of 17 alpha-hydroxylase/17,20-lyase enzyme(s); responsible for the conversion of progesterone to androgen. In summary, results of the present investigation demonstrate that increases in LH activity in the circulation act on two different steroidogenic glands to either enhance or reduce androgen biosynthesis. hCG stimulates luteal secretion of androstenedione and simultaneously inhibits placental production of this steroid.(ABSTRACT TRUNCATED AT 400 WORDS)     

Testicular steroidogenesis in the camel (Camelus dromedarius) during the mating and the nonmating seasons

Enzyme reactions in the camel testis involved in androgen synthesis were studied to determine the factors which account for the low testosterone production during the non-mating season (NMS). Testes excised during the NMS were found to have a relatively high activity of the 3 beta-hydroxysteroid dehydrogenase systems of pregnenolone, 17 alpha-hydroxypregnenolone, and dehydroepiandrosterone, but the 4-ene-17 alpha-hydroxylase and 4-ene-17,20-lyase systems were apparently less active than the 5-ene-17 alpha-hydroxylase and 5-ene-17,20-lyase. On the other hand, testes excised during the mating season (MS) were found to have a relatively high activity of 4-ene-17 alpha-hydroxylase, 4-ene-lyase, and 17 beta-hydroxysteroid oxidoreductase. The 19-hydroxylation and aromatizing activities for testosterone and androstenedione were not detected in testes excised in either season. It is proposed that during the NMS the predominant route of testosterone biosynthesis is pregnenolone leads to 17 alpha-hydroxypregnenolone leads to dehydroepiandrosterone leads to androst-5-en-3 beta, 17 beta-diol leads to testosterone.     

The human kidney is a progesterone-metabolizing and androgen-producing organ

Progesterone (P) is a potent antagonist of the human mineralocorticoid receptor (MR) in vitro. We have previously demonstrated effective downstream metabolism of P in the kidney. This mechanism potentially protects the MR from P action. Here, we have investigated the expression and functional activity of steroidogenic enzymes in human kidney. RT-PCR analysis demonstrated the expression of 5 alpha-reductase type 1, 5 beta-reductase, aldo-keto-reductase (AKR) 1C1, AKR1C2, AKR1C3, 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) type 2, and 17 alpha-hydroxylase/17,20-lyase (P450c17). The presence of 3 beta-HSD type 2 and P450c17 indicated that conversion of pregnenolone to dehydroepiandrosterone (DHEA) and to androstenedione may take place effectively in kidney. To investigate this further, we incubated kidney subcellular fractions with radiolabeled pregnenolone. This resulted in efficient formation of DHEA from pregnenolone, indicating both 17 alpha-hydroxylase and 17,20-lyase activities exerted by P450c17. Radiolabeled DHEA was converted via androstenedione, androstenediol, and testosterone, indicating both 3 beta-HSD type 2 activity and 17 beta-HSD activity. In addition, the conversion of testosterone to 5 alpha-dihydrotestosterone was detectable, indicating 5 alpha-reductase activity. In conclusion, we verified the expression and functional activity of several enzymes involved in downstream metabolism of P and androgen synthesis in human kidney. These findings may be critical to the understanding of water balance during the menstrual cycle and pregnancy and of sex differences in hypertension.     

The cytochrome P450(17) alpha (17 alpha-hydroxylase/C17,20-lyase) activity of the junctional zone of the rat placenta

The cytochrome P450 responsible for androgen synthesis by the placenta during the second half of pregnancy in the rat was studied in intact and hypophysectomized animals. The two activities of P450(17) alpha, 17 alpha-hydroxylase and C17,20-lyase, were limited to the junctional zone. C17,20-Lyase activity was greater with progesterone than with 17-hydroxyprogesterone as substrate. Although the apparent Michaelis constants were similar, progesterone had a higher maximum velocity than 17-hydroxyprogesterone. Regardless of substrate, C17,20-lyase activity was greater with NADPH than with NADH as an electron donor, and there was no additive effect using both cofactors. Administration of human chorionic gonadotrophin (hCG; 10 IU at 09.00 and 21.00 h on days 13 and 14 and at 09.00 h on day 15) to intact females resulted in more than a 50% reduction of enzyme activity when measured on day 15. The same dose of hCG given to hypophysectomized animals with delayed implantation, i.e. pituitary removal on day 3 and implantation induced by oestrone 5 days later, had no effect on placental enzyme activity, but increased that in the ovary. Administration of ovine LH by osmotic minipump (days 11-15) to intact females resulted in abortion in all animals. The same treatment to animals hypophysectomized on day 11 produced abortion in three of four rats; enzyme activity was greatly reduced in the single animal with placentas. In contrast, infusion of LH into hypophysectomized animals with delayed implantation increased placental enzyme activities.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Functional characterization of mutant CYP17 genes isolated from a 17α-hydroxylase/17,20-lyase-deficient patient

CYP17 has a dual enzymatic activity that is necessary for steroid hormone biosynthesis. It catalyzes the 17 alpha-hydroxylation of progesterone or pregnenolone and also removes an acetyl moiety of hydroxy-progesterone or hydroxypregnenolone by its 17,20-lyase activity to produce androstenedione or dehydroepiandrosterone (DHEA), respectively. We previously isolated a compound heterozygous mutant of CYP17 from a Korean female patient: 1-base deletion and 1-base transversion mutation at 1 allele and 3-base deletion mutation at the other allele. Here we tested the functional activities of these 2 mutant CYP17 alleles using a transfection analysis in COS-1 cells with radiolabeled substrates and thin layer chromatography. Both mutant CYP17 genes lost not only 17 alpha-hydroxylation activity, but also 17,20-lyase activity in this assay system. This nonfunctional nature of 2 mutant CYP17 genes explains the clinical manifestation of a patient who had 17 alpha-hydroxylase deficiency.     

Functional characterization of mutant CYP17 genes isolated from a 17 alpha-hydroxylase/17,20-lyase-deficient patient

CYP17 has a dual enzymatic activity that is necessary for steroid hormone biosynthesis. It catalyzes the 17 alpha-hydroxylation of progesterone or pregnenolone and also removes an acetyl moiety of hydroxy-progesterone or hydroxypregnenolone by its 17,20-lyase activity to produce androstenedione or dehydroepiandrosterone (DHEA), respectively. We previously isolated a compound heterozygous mutant of CYP17 from a Korean female patient: 1-base deletion and 1-base transversion mutation at 1 allele and 3-base deletion mutation at the other allele. Here we tested the functional activities of these 2 mutant CYP17 alleles using a transfection analysis in COS-1 cells with radiolabeled substrates and thin layer chromatography. Both mutant CYP17 genes lost not only 17 alpha-hydroxylation activity, but also 17,20-lyase activity in this assay system. This nonfunctional nature of 2 mutant CYP17 genes explains the clinical manifestation of a patient who had 17 alpha-hydroxylase deficiency.     

Differential inhibition of 17alpha-hydroxylase and 17,20-lyase activities by three novel missense CYP17 mutations identified in patients with P450c17 deficiency

The microsomal enzyme cytochrome P450c17 is an important regulator of steroidogenesis. The enzyme has two functions: 17alpha-hydroxylase and 17,20-lyase activities. These functions determine the ability of adrenal glands and gonads to synthesize 17alpha-hydroxylated glucocorticoids (17alpha-hydroxylase activity) and/or sex steroids (17,20-lyase activity). Both enzyme functions depend on correct steroid binding, but it was recently shown that isolated lyase deficiency can also be caused by mutations located in the redox partner interaction domain. In this article we present the clinical history and molecular analysis of two patients with combined 17alpha-hydroxylase/17,20-lyase deficiency and four patients with isolated 17,20-lyase deficiency. In these six patients, four missense CYP17 mutations were identified. Two mutations were located in the steroid-binding domain (F114V and D116V), and the other two mutations were found in the redox partner interaction domain (R347C and R347H). We investigated the activity of these mutated proteins by transfection experiments in COS-1 cells using pregnenolone, progesterone, or their hydroxylated products as a substrate and measuring 17alpha-hydroxylase- and 17,20-lyase-dependent metabolites in the medium. The mutations in the steroid-binding domain (F114V and D116V) of P450c17 caused combined, complete (F114V), or partial (D116V) 17alpha-hydroxylase and 17,20-lyase deficiencies, whereas mutations in the redox partner interaction domain (R347C and R347H) displayed less severe 17alpha-hydroxylase deficiency, but complete 17,20-lyase deficiency. These findings are consistent with the clinical data and support the observation that the redox partner interaction domain is essential for normal 17,20-lyase function of P450c17.     

Molecular modeling of the hamster adrenal P450C17

The cytochrome P450C17 (C17) is the steroidogenic enzyme responsible for the conversion of pregnenolone and progesterone to dehydroepiandrosterone (DHEA) and delta4-androstenedione (AD) respectively. This conversion is achieved by two enzymatic activities, 17alpha-hydroxylase and 17,20-lyase, located at the same active site. In man, the adrenal C17 basically only produces DHEA. We have shown that the hamster adrenal C17 produces DHEA as well as AD. Moreover, the hamster like man produces cortisol as its major glucocorticoid. We can thus compare the hamster and human adrenal C17, and use their differences in order to elaborate a strategy for structure-function studies. We have thus engineered hamster adrenal C17 mutants which possess modified enzymatic activities. We also proceeded to elaborate a three-dimensional model of the hamster C17 to visualise the structural impact of these mutations. This model demonstrates that the mutations created are not localised at the active site, but rather in surrounding regions. These could affect the conformation of the active site, in turn, modulating the 17alpha-hydroxylase and 17,20-lyase activities. For example, the mutation T202N is located next to Val 482 and Val 483 which compose the roof of the active site. This mutation decreased both 17alpha-hydroxylase and 17,20-lyase activities, indicating the importance of the roof of the active site for general functionality of the C17.     

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.     

The in vitro biosynthesis of steroids by the gonad of the cuttlefish (Sepia officinalis L.)

In vitro steroid biosynthesis in the male and female gonads of Sepia officinalis was investigated. Using tritium- and ¹⁴C-labeled precursors, seven enzyme systems were demonstrated: a C_20,22-lyase (cholesterol→pregnenolone), a 3β-hydroxysteroid dehydrogenase-Δ_5?4-isomerase complex (pregnenolone → progesterone; 17α-hydroxypregnenolone → 17α-hydroxyprogesterone; dehydroepiandrosterone → androstenedione), a 17α-hydroxylase (pregnenolone → 17α-hydroxypregnenolone; progesterone → 17α-hydroxyprogesterone), a C_17,20-lyase (17α-hydroxypregnenolone → dehydroepiandrosterone), a 17β-hydroxysteroid dehydrogenase (dehydroepiandrosterone → androstenediol; androstenedione → testosterone; estrone→estradiol-17β), a 20α-ol dehydrogenase (progesterone→20α-dihydroprogesterone), and a 20β-ol dehydrogenase (progesterone→20β-dihydroprogesterone). The yields were rather low (0.01–3%), except when pregnenolone was used (8%). Radioimmunoassay techniques indicate the presence of testosterone in the plasma of the cuttlefish but the absence of estrogens.     

Sites of action of local estradiol feedback mechanism in the frog (Rana esculenta) testis

The direct effect of estradiol on testicular androgen biosynthesis was studied in the frog, Rana esculenta, measuring progesterone, 17 alpha-OH-progesterone, androstenedione, and androgens (T + DHT) in supernatants and testes incubated (6 hr, 15 degrees) with estradiol alone (10(-6) M) or in combination with crude pituitary homogenate (1 pituitary equivalent/tube). Estradiol, either alone or in combination with pituitary homogenate, induced decreases of 17 alpha-OH-progesterone, androstenedione, and androgens but was ineffective in modulating progesterone levels. Pituitary homogenate was effective in inducing a significant increase of androstenedione and androgens but was ineffective in modulating both progesterone and 17 alpha-OH-progesterone production. It is concluded that estradiol acts by decreasing the activity of steroidogenic enzymes starting from 17 alpha-hydroxylase, while pituitary homogenate does not affect the 17 alpha-hydroxylase activity, but it acts starting from 17,20-lyase.     

Selective stimulation of luteal androgen biosynthesis by luteinizing hormone: comparison of hormonal regulation of P45017 alpha activity in corpora lutea and follicles

Recent investigations have indicated that luteal cells of pregnant rats become capable of synthesizing androgen and estradiol when stimulated by sustained low levels of LH/hCG. In this investigation we sought 1) to determine whether hCG causes the induction/activation of the luteal enzymes responsible for the conversion of progesterone to estradiol, 2) to investigate the time course of hCG action, and 3) to compare the effect of hCG on luteal and follicular P45017 alpha activities. To determine first the minimum stimulatory dose of hCG, pregnant rats were treated with 0, 1.5, 3, 9, or 30 IU hCG twice on days 12 and 13 and once on day 14. Two hours after the last injection, rats were bled from the ovarian vein, and corpora lutea were isolated and incubated for the determination of in vitro steroid production. Exposure of rats to twice daily injections of 1.5 IU hCG caused a dramatic increase in the ovarian secretion and luteal production of both testosterone and estradiol. However, an inverse dose-related response was observed with higher doses of hCG. To determine the time course of hCG action, pregnant rats were injected with a single dose of 3 IU hCG, and steroid production was determined at different times thereafter. There was no increase in either in vivo or in vitro testosterone and estradiol production until 12 h after hCG administration, at which time a dramatic increase in the secretion of both steroids was observed. hCG administration did not affect the capacity of luteal cells to secrete progesterone, nor did it enhance aromatase activity. It did, however, increase P45017 alpha activities; lyase and hydroxylase activities were stimulated 5- and 1.7-fold, respectively. To compare the effects of hCG on luteal and follicular P45017 alpha, both corpora lutea and follicles were isolated from ovaries of pregnant rats treated with either 1.5 IU hCG or vehicle between days 12-14. In vivo hCG administration caused at least a 1000-fold increase in the specific activity of follicular 17 alpha-hydroxylase and 17,20-lyase. The hCG-induced increase in the specific activities of both hydroxylase and lyase in the follicle far exceeded that in the corpora lutea. However, total lyase and hydroxylase activities in each follicle were only 3- and 5.5-fold higher, respectively, than those in the corpus luteum. In summary, the results of the present investigation have revealed that hCG stimulation of luteal estradiol production is due to a selective effect of this gonadotropin on P45017 alpha.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ovine placental steroid 17 alpha-hydroxylase/C-17,20-lyase, aromatase and sulphatase in dexamethasone-induced and natural parturition

Parturition in the sheep is preceded by an abrupt alteration in placental steroid metabolism causing a shift from progesterone to oestrogen production. This change is believed to be a consequence of the preparatum rise in cortisol in the fetal circulation and involves increases in activities of the enzymes steroid 17 alpha-hydroxylase (cytochrome P-450(17) alpha), steroid C-17,20-lyase, and possibly aromatase and steroid sulphatase. The activity levels have been determined of steroid 17 alpha-hydroxylase, aromatase and steroid sulphatase in placental microsomes in late pregnancy, dexamethasone-induced labour and in natural labour at term. Over the gestational period of 118-140 days, basal levels of placental aromatase were relatively constant (mean value (+/- S.E.M.) of 5.6 +/- 0.5 pmol/min per mg microsomal protein (n = 10]. Pregnenolone and progesterone 17 alpha-hydroxylase activities were undetectable (less than 0.5 pmol/min per mg microsomal protein (n = 7]. In six animals in labour induced with infusion of dexamethasone into the fetus, placental aromatase activity increased to a value of 14.0 +/- 1.0 pmol/min per mg protein; placental pregnenolone 17 alpha-hydroxylase, measured in four of the animals, also increased to 453 +/- 77 pmol/min per mg microsomal protein. In five animals in natural spontaneous labour with vaginal delivery, aromatase activity was 26.7 +/- 5.2 pmol/min per mg microsomal protein and pregnenolone 17 alpha-hydroxylase activity was 141 +/- 14 pmol/min per mg microsomal protein. Steroid sulphatase activity was barely detectable (less than 1.5 pmol/min per mg microsomal protein) during late pregnancy, dexamethasone-induced labour or natural parturition.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

LH-induced inhibition of follicular androgen formation requires intact steroidogenesis

LH exerts a biphasic effect on rat pre-ovulatory follicular steroidogenesis: an initial (1–4h) overall stimulation followed by a later (4–6 h) occurring inhibition of androgen synthesis. Because exogenous steroids may inhibit androgen formation, we investigated whether the steroids produced initially in response to LH are involved in the late inhibition of androgen synthesis. Isolated pre-ovulatory rat follicles were incubated for 6 h with and without ovine LH and 1 of 3 inhibitors of steroidogenesis (aminoglutethimide, cyanoketone, Su 10603). Accumulation of androstenedione and testosterone in a subsequent 2-h incubation in the presence of exogenous 17-hydroxyprogesterone was measured. LH treatment alone caused inhibition of apparent 17,20-lyase activity. The inhibitors had no effect on basal 17,20-lyase activity but were able to prevent the LH-induced inhibition of this enzymic activity. The results suggest that the physiological decline in pre-ovulatory androgen formation may in part be mediated by local action of follicular steroids.     

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.     

The in vitro biosynthesis of steroids by the gonad of the mussel (Mytilus edulis)

In vitro steroid biosynthesis in the male and female gonad of Mytilus edulis was investigated. Using tritium and ¹⁴C-labeled precursors, four enzyme systems were demonstrated: a 3β-hydroxysteroid dehydrogenase-Δ_5,4-isomerase comples (pregnenolone→progesterone; 17α-hydroxypregnenolone→17α-hydroxyprogesterone; dehydroepiandrosterone→androstenedione), a C_17,20-lyase (17α-hydroxypregnenolone→dehydroepiandrosterone; 17α-hydroxyprogesterone→androstenedione), a 17β-hydroxysteroid dehydrogenase (dehydroepiandrosterone→androstenediol; androstenedioneαtestosterone; estradiol-17βαestrone) and a 5α-reductase (testosterone→5α-dihydrotestosterone). The yields were very low (0.2–1%) except when estrogens were used (10–50%). Radioimmunoassay techniques indicate the presence of testosterone and possibly estrogens in the gonad of the mussel at two different stages in its gametogenic cycle.     

Biosynthetic pathways of testosterone and estradiol-17 beta in slices of the embryonic ovary and testis of the chicken (Gallus domesticus)

To elucidate synthetic pathways of testosterone and estradiol-17 beta in embryonic gonads of the chicken, metabolism of various 14C-labeled steroids in slices of the left ovaries and paired testes of 15- and 9-day-old chicken embryos was examined. (1) Fifteen-day-old chicken embryos: From pregnenolone, more 17 alpha-hydroxypregnenolone was produced than progesterone in the ovary, while more progesterone was produced than 17 alpha-hydroxypregnenolone in the testis. From 17 alpha-hydroxypregnenolone, however, only dehydroepiandrosterone was detected as a product in both gonads. Dehydroepiandrosterone was converted mainly into androstenedione and its 5 beta-reduced derivatives by both gonads. Progesterone was converted into 5 beta-pregnane-3,20-dione more than into 17 alpha-hydroxyprogesterone by both gonads. Both gonads metabolized 17 alpha-hydroxyprogesterone, androstenedione, and testosterone predominantly into their corresponding 5 beta-reduced steroids, while production of androstenedione from 17 alpha-hydroxyprogesterone and of testosterone from androstenedione was limited. Estradiol-17 beta was produced from androstenedione and testosterone only by the ovary. (2) Nine-day-old chicken embryos: From pregnenolone, production of progesterone and 17 alpha-hydroxypregnenolone was similar in the ovary. On the other hand, in the testis, more progesterone was produced than 17 alpha-hydroxypregnenolone from pregnenolone. For delta 4-3-oxo steroids, strong activity of 5 beta-reductase was demonstrated in both gonads. From these results, both delta 4- and delta 5-pathways are involved in the formation of testosterone and then finally of estradiol-17 beta by the embryonic gonads of the chicken, and relative preference for the pathway seems to depend on sexes and embryonic ages. In addition, it is suggested that steroidogenesis in these embryonic gonads is characterized by marked activity of 5 beta-reductase, irrespective of sexes or ages.     

EFFECTS OF HIGH DOSE KETOCONAZOLE THERAPY ON THE MAIN PLASMA TESTICULAR AND ADRENAL STEROIDS IN PREVIOUSLY UNTREATED PROSTATIC CANCER PATIENTS

In vitro, ketoconazole has been shown to block testicular and adrenal 17,20-lyase, which converts progestins to androgens. At higher concentrations, it also inhibits 11 beta-hydroxylase, 20,22-desmolase and 17 alpha-hydroxylase. To determine the differential hormonal effects of a 2-week ketoconazole high-dose therapy, the plasma levels of 10 major androgens, gluco- and mineralocorticoids were measured in 14 previously untreated patients with metastatic prostate cancer. Within 24 h, plasma testosterone fell from 14.6 +/- 1.4 nmol/l (mean +/- SEM) to 3.7 +/- 0.7 nmol/l. Thereafter, it decreased to about 2.5 nmol/l and remained at that level. Plasma androstenedione and dehydroepiandrosterone decreased more gradually, respectively from 3.1 +/- 0.4 nmol/l to 0.64 +/- 0.17 nmol/l and from 6.6 +/- 1.0 nmol/l to 2.82 +/- 0.55 nmol/l (on day 14). In contrast, 17 alpha-hydroxyprogesterone and progesterone rose respectively 2- and 5-fold. Plasma cortisol and aldosterone levels remained unchanged whereas 11-deoxycorticosterone and 11-deoxycortisol rose by factors of 14 and 6.7 respectively. Plasma corticosterone also increased, but to a much lesser extent (3-fold). These results demonstrate that ketoconazole high dose therapy blocks mainly the 17,20-lyase of both adrenal and testis. In addition it inhibits mitochondrial 11 beta-hydroxylase to a lesser extent. The inhibition of 20,22-desmolase also seems to be of little clinical relevance. However, since clinical or laboratory symptoms suggestive of hypo-adrenalism have been reported in a small minority of patients, replacement therapy should be considered in such cases.