Testicular steroidogenesis in the baboon Papio anubis.

We recently reported that the baboon testis converts pregnenolone to testosterone through the delta-4 pathway. The present studies were to determine the metabolism of intermediates of the delta-4 and delta-5 pathway by the baboon testis. Fragments (50 mg) were incubated for 3 hr with 10 muCi of the following tritium-labelled substrates: pregnenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, or testosterone. Pregnenolone was converted to testosterone primarily through the delta-4 pathway, with accumulation of progesterone, 17-hydroxyprogesterone and 20alpha-dihydroprogesterone as predominant intermediates. Similar results were obtained in progesterone incubations. 17-hydroxyprogesterone was not efficiently metabolized by the fragments, while 17-hydroxypregnenolone and dehydroepiandrosterone were efficiently converted into testosterone and androstenedione. Androstenedione was metabolized primarily to testosterone, while testosterone was not a suitable substrate. Some 5alpha-androstanediol was identified in each incubate. These results suggest that although testosterone is formed from pregnenolone through the delta-4 pathway, the delta-5 intermediates are more suitable substrates for testosterone synthesis in the baboon testis.     

An adrenocortical tumor secreting weak mineralocorticoids

An adrenocortical carcinoma (15.5 g) secreting excessive amounts of steroids with weak mineralocorticoid activity in a 25-year-old woman was studied with particular reference to its in vivo and in vitro secretions of steroids. Severe hypertension, occasional low serum potassium and suppressed PRA were the major clinical findings, and were improved with removal of the tumor. In the preoperative stage, plasma levels of 11-deoxycorticosterone, 18-hydroxy-11-deoxycorticosterone, corticosterone and 18-hydroxycorticosterone were all increased. However, the plasma level of aldosterone was repeatedly normal. Although plasma levels of pregnenolone, 17-hydroxypregnenolone, progesterone and 17-hydroxyprogesterone were very high, those of other late step steroids, i.e. 11-deoxycortisol, cortisol, dehydroepiandrosterone, androstenedione and testosterone were almost normal. From these findings, a major etiological role of weak mineralocorticoids such as 11-deoxycorticosterone, 18-hydroxycorticosterone and corticosterone in her hypertension was suggested. Pregnenolone and 17-hydroxypregnenolone in tumor tissue were increased, but 11-deoxycorticosterone, corticosterone, aldosterone, cortisol and adrenal androgens such as dehydroepiandrosterone, androstenedione and testosterone were below normal or low normal. In vitro production of 11-deoxycorticosterone, aldosterone or cortisol by the tumor tissue slices was very low and scarcely responded to synthetic ACTH.     

Testicular steroidogenesis in the rhesus monkey (Macaca mulatta.

Studies were undertaken to investigate testicular steroidogenesis in the Rhesus monkey Macaca mulatta. Testicular fragments (50 mg) were incubated for 3 hr with pregnenolone-7-3H or with progesterone-7-3H. The major metabolite of pregnenolone was progesterone (70.1%), with a lesser conversion to 17-hydroxyprogesterone (1.6%), androstenedione (3.3%), and testosterone (7.2%). The delta-5 intermediates 17-hydroxypregnenolone (4.6%) and dehydroepiandrosterone (8.6%) were also identified in the pregnenolone incubates. A majority of the progesterone substrate was not metabolized by the testicular fragments (80.1%), while some conversion to 17-hydroxyprogesterone (3.4%), androstenedione (4.8%), and testosterone (11.7%) occurred in the incubates. These results suggest that testicular fragments from the Rhesus monkey may convert pregnenolone to testosterone through both the delta-4 and the delta-5 pathways.     

Effect of acute ACTH administration on plasma steroid levels in the dog

Production of adrenal steroids in intact and castrated dogs is stimulated acutely by ACTH. While the increase in plasma cortisol, 17-hydroxypregnenolone and 17-hydroxyprogesterone is not affected by castration, the increment of dehydroepiandrosterone is totally abolished. On the other hand, administration of 17-hydroxypregnenolone in adrenalectomized dogs caused an increase in plasma C-19 steroids such as dehydroepiandrosterone, androstenedione and testosterone indicating that this C-21 progestin in plasma is rapidly converted. The site of this conversion is likely the testis. Furthermore, acute hCG administration in adrenalectomized dogs resulted in a marked increase in the levels of plasma 17-hydroxypregnenolone and dehydroepiandrosterone. However, our data show an ACTH-induced rise in 5-androstene-3 beta. 17 beta-diol in intact and castrated dogs, thus suggesting that this steroid is a good parameter to assess in the stimulation of adrenal steroidogenesis by ACTH.     

The conversion of pregnenolone-7alpha-3H and progesterone-4-14C to estrogens by corpora lutea of menstrual cycles and pregnancy.

The metabolism of pregnenolone-7alpha-3H and progesterone-4-14C by human corpora lutea tissue of menstrual cycles and pregnancy was studied. In the incubations, equimolar mixtures of pregnenolone-7alpha-3H and progesterone-4-14C were used as substrates. Three corpora lutea of cycles were used as minced tissue. From those corpora lutea progesterone, 17-hydroxyprogesterone and androstenedione were formed, although no estrogens were formed. One corpus luteum of cycle and one corpus luteum of pregnancy were used as homogenated tissue, and those formed estrone and estradiol as well as the same three delta4-metabolites. The corpus luteum of cycle also formed testosterone. All metabolites including estrogens showed the lower 3H to 14C ratio than the starting ratio. 17-hydroxypregnenolone in only one corpus luteum, and no delta5-metabolites in the other four corpus luteum were identified. It is therefore proposed that the major pathway for estrogen formation in human corpus luteum is pregnenolone yields progesterone yields 17-hydroxyprogesterone yields androstenedione (or testosterone) yields estrone and estradiol.     

Steroid hormone formation in human ovarian follicles in vitro.

Ovarian follicles of 5 to 15 mm in diameter were isolated from 45 ovaries of 34 patients in the follicular and luteal phases of the cycle. Three experiments were done. In the first, follicles were minced and incubated in Krebs-Ringer bicarbonate buffer containing 1 to 2muCi of testosterone-4-14C in the presence or absence of 100 IU human chorionic gonadotropan (hCG). In the second, minced follicles were incubated with 100 muCi of sodium acetate-I-14C under identical conditions. In the third, ten follicles from a single patient in the late proliferative stage of endometrial dating were cut in halves and incubated with 100 muCi of acetate-I-14C under identical conditions. The minced follicle preparation was capable of aromatizing testosterone-4-14C into radioactive estrone and estradiol in significant amounts. Incorporation of radioactive acetate into pregenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, testosterone, estradiol and estrone was assessed by reverse dilution analysis with recrystallization to constant specific activity. The major radioactive products formed were androstenedione and 17-hydroxyprogesterone in the latter two experiments. Dehydroepiandrosterone was one of the major steroids in the second experiment. The minor products were testosterone, progesterone and pregnenolone. Smaller, but definite incorporations of radioactive acetate into estradiol and estrone occurred in the second experiment. On histological examination, the follicles were characterized by atretic changes. This distribution pattern of radioactive acetate among the steroids was considered to represent the steroidogenic profile of unstimulated or atretic follicles.     

Comparison of serum steroid responses to a single injection of hCG in man and rat

The responses of peripheral serum steroids to a single injection of hCG (80 IU/kg b wt) were compared in adult male rats and humans. Before hCG, the quantitatively dominating steroids were dehydroepiandrosterone, testosterone and 17-hydroxypregnenolone in the men, and testosterone and progesterone in the rats. One hour after hCG the concentrations of testosterone and all its precursors measured except for pregnenolone were significantly elevated in the rat serum, whereas a clear rapid response was not observed in the men. Transient blockade of C21 steroid side-chain cleavage was seen in both species at about 24-36 h after hCG, which occurred at the same time as the maximum concentration of estradiol in the men. No changes in rat serum estradiol concentrations were observed. Both species showed a secondary stimulation of testosterone and androstenedione formation at around 3 days. Our findings are compatible with the concept that the main difference in the gonadotropin-stimulated steroidogenesis in man and rat is the magnitude of the rapid steroidogenic response to hCG, which is very small in man and indicates smaller supply or lesser metabolism of mitochondrial cholesterol in human testis.     

Effect of in vitro ketoconazole on steroid production in rat testis

In an attempt to confirm where in the testosterone (T) biosynthetic pathway of the rat testis ketoconazole (KTZ) inhibits T production, rat testicular mince was incubated with either 10 micrograms/ml or 100 micrograms/ml KTZ in the presence and absence of hCG (1 IU), and intratesticular pregnenolone (delta 5P), progesterone (P), 17-alpha-hydroxyprogesterone (17 alpha-HP), androstenedione (A) and testosterone (T) were assayed. In the absence of hCG, 10 micrograms/ml KTZ was sufficient to reduce intratesticular T by 80%. At this concentration of KTZ, intratesticular 17 alpha-HP (ng/g testis, mean +/- SEM) increased from 0.3 +/- 0.1 to 1.3 +/- 0.2 (p less than 0.0025), whereas intratesticular A decreased from 84 +/- 7 to 17 +/- 1 (p less than 0.005). KTZ did not inhibit the conversion of P to 17 alpha-HP. From these data it was concluded that KTZ has its inhibitory effect on testosterone biosynthesis in the rat testis primarily at the step catalyzed by the 17,20 desmolase enzyme.     

Subnormal pubertal increases of serum androgens in Turner's syndrome

60 patients (139 blood specimens) with Turner's syndrome were investigated in order to obtain information concerning the origin of the increments of androgens during puberty. The concentrations of serum FSH, LH, estradiol, testosterone, 5 alpha-dihydrotestosterone, dehydroepiandrosterone, progesterone, 17-hydroxyprogesterone and pregnenolone in patients less than 10 years old were identical to those previously found in normal healthy girls of the same age. Hence, in adrenarche the early increase of androgen secretion is independent of gonadal hormone secretion. The later increases in serum testosterone and androstenedione in our patients were very small, and the age of 15 years, their concentrations were 50 and 60%, respectively, of the corresponding levels in normal girls of the same age. After 13 years of age, the mean serum dehydroepiandrosterone concentration was also slightly, but significantly (20-30%), lower than in normal girls of the same age. It is concluded that the ovaries are responsible for most of the pubertal rises in circulating testosterone and androstenedione, and possibly for a small part of the late pubertal rise in dehydroepiandrosterone.     

Effects of restraint stress on plasma LH and testosterone concentrations, Leydig cell LH/hCG receptors, and in vitro testicular steroidogenesis in adult rats

We examined the effect of restraint stress (3 hr) on plasma LH and testosterone levels, on the Leydig cell LH/hCG receptor, and on the activity of enzymes in the testicular steroidogenic pathway of the adult rat. Restraint stress caused a 47% reduction in plasma testosterone concentrations, but had no effect on plasma LH levels. The binding capacity and affinity of Leydig cell LH/hCG receptors were not affected by restraint. Stress did not affect the testicular activity of 20,22 desmolase or 3 beta-hydroxysteroid dehydrogenase, but testicular interstitial cells of stressed rats incubated in vitro with progesterone as a substrate produced more 17 alpha-hydroxyprogesterone but less testosterone than control cells, and when incubated with 17 alpha-hydroxypregnenolone, produced 39% less androstenedione and 40% less testosterone than control cells. These results suggest that restraint stress inhibited 17,20 desmolase but not 17 alpha-hydroxylase activity. When the delta 4 pathway was blocked with cyanoketone (3 beta-HSD inhibitor), stress did not alter the production of pregnenolone or 17 alpha-hydroxypregnenolone, but the production of dehydroepiandrosterone by cells from stressed rats was subnormal, suggesting again a reduction of 17,20 desmolase activity. The data suggest that a major site of the inhibitory action of restraint stress on testicular steroidogenesis is the 17,20 desmolase step. The disruption of androgen production by restraint appears to be LH independent since stress did not affect plasma LH levels, the binding capacity or affinity of LH/hCG receptors, or the activity of 20,22 desmolase.     

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.     

Inhibition of testosterone biosynthesis by ethanol: relation to the pregnenolone-to-testosterone pathway

The concentrations of metabolites in the pregnenolone → testosterone pathway were determined in freezestopped testes in control rats and during ethanol intoxication (2 h after injection of 1.5 $\text{g ethanol}\text{kg}$ body wt). Ethanol lowered the mean testicular concentrations of testosterone (by 63–74%), androstenedione (49–81%), 17-hydroxyprogesterone (60–76%), progesterone (29–67%) and pregnenolone (12–25%). 4-Methylpyrazole had no effect on the ethanol-induced changes. The present results reveal no inhibition at the 17-hydroxyprogesterone → androstenedione → testosterone steps, but do not exclude inhibition before the step yielding pregnenolone and at the pregnenolone → progesterone → 17-hydroxyprogesterone steps.     

Plasma sex hormone concentrations during the reproductive cycle in the male lizard, Podarcis s. sicula

Progesterone, 17-hydroxyprogesterone, androstenedione, 5 alpha-dihydrotestosterone, dehydroepiandrosterone, testosterone and oestradiol concentrations in the plasma were measured by simultaneous radioimmunoassay in males of the lizard Podarcis s. sicula. Hormonal determinations were performed at monthly intervals from January to December (except for August). Testosterone and androstenedione reached peak values of 174.8 ng/ml and 21.4 ng/ml in the mating season (spring) and then testosterone fell abruptly to 5.9 ng/ml in June remaining at this level during hibernation when dehydroepiandrosterone (DHA) reached a maximal level of 28.5 +/- 9.3 ng/ml. Castration resulted in a marked decrease of testosterone, androstenedione, dihydrotestosterone and DHA values, with DHA being significantly lowered only during the winter season. In castrated animals, however, testosterone and androstenedione persisted conspicuously in the plasma during the breeding period, suggesting that adrenal sex steroid output may change during the annual reproductive cycle. In intact animals, progesterone and oestradiol exhibited peak values during the refractory period after the mating season. We suggest a probable role of oestradiol in the induction of the refractory period in this lizard.     

Partial deficiency in 17-ketosteroid reductase presenting as gynecomastia

We report a 14 year-old male with severe, long-lasting gynecomastia. Baseline serum androstenedione levels were elevated compared to testosterone levels (330 ng/dl vs 28 ng/dl). In order to evaluate testosterone biosynthesis by this patient in more detail, androstenedione, testosterone, dehydroepiandrosterone (DHEA) and estradiol responses to a single dose of hCG were measured. The responses observed were different from those reported in normal males in two respects: 1) there was no immediate rise in testosterone two to four hours after the injection of hCG, and 2) levels of androstenedione and estradiol at 24, 36 and 48 hours after injection were much higher than expected. We postulate that a partial defect in testicular 17-ketosteroid reductase activity was responsible for the abnormal androstenedione to testosterone ratio in our patient. This, in turn, lead to an increased peripheral synthesis of estrogens and marked gynecomastia.     

17,20-desmolase deficiency in a female newborn, paradoxically virilized in utero

The patient was born with ambiguous genitalia (stade III of Prader). The karyotype revealed a normal female genotype. A defect in 21-hydroxylase, at first suspected, was denied by the hormonal studies. Indeed, extremely high levels of pregnenolone, pregnenolone sulfate, progesterone were found in association with low plasma levels of delta 4-androstenedione, testosterone, dehydroepiandrosterone and its sulfate, while cortisol 17OH-progesterone and ACTH levels and plasma renin activity were normal. The hormonal pattern was thus consistent with 17,20-desmolase deficiency. The dynamic studies further supported this contention: all the progestagens rose further after ACTH stimulation and were suppressed by dexamethasone. Meanwhile, all androgens failed to rise after ACTH: the responses of cortisol were normal. The in utero virilization of the female fetus was not understood until an history of virilization was allegedly found in the mother (luteoma of pregnancy). This is the first case of 17-20 desmolase defect recognized in a female newborn. This child, born with ambiguous genitalia had presented the concurrence of two very rare conditions. The in utero virilization of maternal origin enabled us to make the diagnosis of the 17-20 desmolase defect, which otherwise would have been ignored in a XX subject in the neonatal period because it would obviously be unsymptomatic at this age.     

Adrenal steroidogenesis in the guinea pig: effects of androgens.

In humans, the onset of adrenache has been found to occur with the appearance of the zona reticularis, the inner zone of the adrenal cortex. Since an increase in the volume of adrenal cortex during maturation in the guinea pig has been associated with the growth of the zona reticularis, we were interested in investigating the changes in adrenal steroidogenesis during maturation in this species. In addition, the effect of androgens on adrenal steroidogenesis was studied. We demonstrated that between 1 and 10 weeks of age, a period of maximal growth of the adrenals in the guinea pig, there is a decrease in the concentrations of adrenal pregnenolone, cortisol, dehydroepiandrosterone, testosterone, androstenedione, and 11 beta-hydroxyandrostenedione, suggesting lower steroid production by the guinea pig adrenals. In plasma, we observed that the concentration of 11 beta-hydroxyandrostenedione (the sole C19 steroid present after castration) remained unchanged during maturation, while cortisol and corticosterone were lower between 1 and 4 weeks of age. Although castration as well as the administration of the antiandrogen flutamide had no effect on adrenal steroidogenesis, dihydrotestosterone caused an inhibition of cortisol and corticosterone levels in the adrenals while the concentrations of progestins (namely, pregnenolone, 17-hydroxypregnenolone, progesterone, and 17-hydroxyprogesterone) tended to increase in the adrenals, thus suggesting that dihydrotestosterone induces a blockade in the steroidogenic pathway.(ABSTRACT TRUNCATED AT 250 WORDS)     

Male pseudohermaphroditism due to testicular 17-ketosteroid reductase deficiency.

A new case of testicular 17 ketosteroid reductase (17 KSR) deficiency without gynecomastia was investigated. Delta4 androstenedione (15.6 ng/ml) was ten times the normal range, unchanged after dexamethasone administration. In contrast, plasma testosterone (4.1 ng/ml) was in the low normal male range and plasma dehydroepiandrosterone (4.2 ng/ml) was normal. Plasma luteinizing hormone and follicle-stimulating hormone were increased (162 and 470 ng/ml LER 907 respectively). After adrenal suppression and human chorionic gonadotropin stimulation, the increase of delta4 androstenedione was in contrast with the inertia of testosterone. In spermatic venous plasma delta4 androstenedione level (293.2 ng/ml) was very high and testosterone level (7.1 ng/ml) a hundred times below the normal mean. Plasma estrone (124 pg/ml) was increased and estradiol (22 pg/ml) was normal. In spermatic venous plasma estrone was elevated and estradiol very low (1380 and 32 pg/ml respectively). It is the third case of 17 KSR deficiency where the lack of E2 increase explains the absence of gynecomastia.     

Testosterone, androstenedione, progesterone and 17 alpha-hydroxyprogesterone in plasma and testes of immature rats under basal conditions and after hCG stimulation. Effect of bilateral cryptorchidism

Rats were made bilaterally cryptorchid at 21 days of age; sham-operated rats were used as controls. At 35 days, the animals were injected i.m. with saline or with 10 IU hCG. Progesterone, 17-hydroxyprogesterone, androstenedione and testosterone were measured in both testes and plasma under basal conditions and 2, 4, 8, 12, 24 and 72 h respectively after injection. The plasma levels and intratesticular contents of the steroids were generally lower in cryptorchid rats. The patterns of the steroid response to hCG were similar in both groups: in the testes and in the plasma, they increased acutely following hCG injection (except testicular androstenedione), then, after 72 h, returned to normal values in the plasma but remained higher than the basal values in the testes. These results suggest that there are no gross abnormalities in the testicular steroidogenic pathways and that the mechanism of action of hCG on the Leydig cells is unaltered in bilaterally cryptorchid immature rats.     

Mild adrenal 3 beta-hydroxysteroid dehydrogenase deficiency with hyperaldosteronism

The patient was admitted to our hospital at 19 and again at 22-yr of age for hirsutism and hypertension. Her baseline and ACTH-stimulated plasma 17-hydroxy pregnenolone, dehydroepiandrosterone and dehydroepiandrosterone sulfate were increased whereas plasma 17-hydroxy progesterone and androstenedione were normal and responded poorly to ACTH. Plasma deoxycorticosterone, corticosterone and cortisol baseline levels were normal, and they responded normally to ACTH. The plasma aldosterone concentration (PAC) was always high and responded well to ACTH, angiotensin III and furosemide-upright stimulation. However, plasma renin activity (PRA) was normal or slightly high, and responded normally to furosemide-upright stimulation and fluorohydrocortisone suppression. Dexamethasone (2 mg/day) for 1-2 weeks suppressed the androgens, cortisol and corticosterone levels. PRA and PAC were suppressed temporally, but PRA returned to normal and PAC to be a high level after 2 weeks of dexamethasone administration. Blood pressure was also reduced temporally but returned to a high level after 2 weeks of dexamethasone. These results indicate that primary aldosteronism and dexamethasone-suppressible hyperaldosteronism were not likely to be present, and unknown aldosterone stimulating factors which potentiated the action of endogenous angiotensin II or ACTH might be responsible for the hyperaldosteronism in this patient. We conclude that this patient had a mild and non-salt losing 3 beta-HSD deficiency in the zona reticularis with normal fasciculata and high glomerulosa function.     

Studies on the metabolism of steroid hormones in a virilizing adrenal cortex adenoma.

Slices of an adreno-cortical adenoma which had been obtained at operation from an 11-year-old girl with clinical signs of virilism were incubated with each of the following steroids: [1,2-3H]progesterone, [4-14C]pregnenolone, [1,2-3H]testosterone, [4-14C]androstenedione and [7-3H]dehydroepiandrosterone, respectively. Isolation and identification of the free radioactive metabolites were achieved by gel column chromatography on Sephadex LH-20, thin-layer chromatography, radio gas chromatography and isotope dilution. After incubation of progesterone, the following metabolites were identified: 11beta-hydroxyprogesterone, 16alpha-hydroxyprogesterone, 17alpha-hydroxyprogesterone, 21-deoxycortisol, corticosterone and cortisol. Pregnenolone was metabolized to 17alpha-hydroxypregnenolone, progesterone, dehydroepiandrosterone, androstenedione and 11beta-hydroxyandrostenedione. When testosterone was used as substrate, 11beta-hydroxytestosterone, androstenedione and 11beta-hydroxyandrostenedione were found as metabolites, whereas androstenedione was metabolized to testosterone and 11beta-hydroxyandrostenedione. After incubation of dehydroepiandrosterone, only androstenedione and 11beta-hydroxyandrostenedione were isolated and identified. From these results, it appears that cortisol was formed in the adenoma tissue via 21-deoxycortisol and corticosterone. Delta4-3oxo steroids of the C19-series arose exclusively from pregnenolone via 17alpha-hydroxypregnenolone and dehydroepiandrosterone, and not from progesterone and 17alpha-hydroxyprogesterone. Calculated on the amounts of metabolites formed, the highest enzyme activities were those of the 11beta-hydroxylase and the 17alpha-hydroxylase. It is interesting to note that only traces of testosterone were detected after incubation of androstenedione, whereas testosterone yielded large amounts of androstenedione.