We describe the third report testosterone-producing, virilizing, adrenal Leydig cell adenoma, which was identified in a oophorectomized postmenopausal female patient. Light- and electron-microscopy demonstrated typical Leydig cell differentiation, including numerous intracytoplasmic and intranuclear Reinke crystals and rice-like bodies (elementary tubular inclusions). Testosterone production by the adenoma was demonstrated by the immunoperoxidase technique. We propose that adrenal Leydig cell adenomas arise as a result of ovarian gonadal stromal metaplasia associated with elevated follicle-stimulating hormone and luteinizing hormone in the postmenopausal female patient. Adrenal Leydig cell lesions must be considered in the virilized woman with elevated testosterone levels and normal levels of the adrenal androgens and their <em>17</em>-<em>ketosteroid</em> metabolites.

A 15-year-old girl who presented with primary amenorrhea and virilization had an adrenocortical adenoma that secreted predominantly testosterone. To our knowledge, she is the first peripubertal and second youngest patient with a testosterone-secreting adrenal tumor described. Serum dehydroepiandrosterone sulfate and urinary <em>17</em>-<em>ketosteroid</em> and <em>17</em>-hydroxycorticosteroid levels were normal. A tumor was located by a computed tomographic (CT) scan and by uptake of 6-beta-[75Se] selenomethylnorcholesterol. Microscopic examination of the tumor showed typical features of an adrenocortical adenoma with no histologic features characteristic of Leydig cells. Postoperatively, her hirsutism regressed, she rapidly went through puberty, and regular monthly menstruation started four months later. Finding the source of testosterone in a virilized patient can be difficult. Eleven of the 14 previously described patients with testosterone-secreting adrenal tumors initially underwent misdirected surgery on the ovaries. Review of these cases revealed that results of hormone stimulation and suppression tests are unreliable and that these tumors are usually large. Therefore, CT scanning of the adrenal glands is recommended in all patients suspected of having a testosterone-secreting tumor. If the adrenal glands on CT scan are normal, then surgery directed at the ovaries can be undertaken. Adrenal and ovarian vein catheterization is rarely necessary.

Simultaneous serum concentrations of dehydroepiandrosterone sulfate (DHEA-S) and <em>17</em>-hydroxyprogesterone (<em>17</em>-OHP) were compared with urinary <em>17</em>-<em>ketosteroid</em> (<em>17</em>-KS) and pregnanetriol (PT) excretion during therapy in 18 prepubertal patients with the 21-hydroxylase deficiency form of congenital adrenal hyperplasia (CAH). Patients were classified into those in good, poor, or questionable control on the basis of clinical examination, skeletal age, and <em>17</em>-KS and PT excretion. During therapy, use of serum steroid concentrations was found to be nearly as accurate in judging adequacy of control as use of urine steroid concentrations. Of 34 evaluations, a definite assessment of adequacy of control could be arrived at 25 times using urinary values and 22 times using both serum DHEA-S and <em>17</em>-OHP concentrations. DHEA-S concentration responded sluggishly when treatment was not adequate, being greater than 100 microgram/dl only in patients significantly undertreated. It was never elevated in well-controlled patients. Mid-afternoon <em>17</em>-OHP concentrations were less than 200 ng/dl in well-controlled patients but readily escaped suppression and could not be used to differentiate poor from borderline control or from temporary noncompliance. Therefore, an increases DHEA-S concentration indicated poor control and a suppressed <em>17</em>-OHP concentration indicated good control. The combination of normal DHEA-S level with elevated <em>17</em>-OHP level, however, did not permit exact evaluation of the degree of control. Of significance is that not all patients with CAH present with an elevated DHEA-S concentration, and only in those in whom an elevated level has been documented can DHEA-S level be used as an index of control during therapy.

The preparation of novel steroidal heterocycles containing the 7-aryl-substituted 1,2,4-triazolo[1,5-a]pyrimidine moiety fused to the 16,<em>17</em>-positions of the steroid nucleus is described. The Aldol reaction of 4-aza-androst-3,<em>17</em>-dione (1a) and dehydroepiandrosterone (DHEA, 1b) with aromatic aldehydes was catalyzed by KF/Al(2)O(3) to give the corresponding 3-oxo-4-aza-5α- and 3β-hydroxy-5-en-16-arylidene-<em>17</em>-<em>ketosteroids</em> (2a-r). Subsequently, the intermediates 2a-r reacted with dinucleophilic 3-amino-1,2,4-triazole in presence of t-BuOK to afford the title compounds (3a-r). All the synthesized heterosteroids are new and are currently being evaluated for their biological activities.

The enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta HSD) is essential for the biosynthesis of all steroid hormones. The enzyme catalyzes the conversion of delta 5-3 beta-hydroxysteroids to delta 4-3-<em>ketosteroids</em>. We report the isolation of a novel mouse 3 beta HSD cDNA, 3 beta HSD IV, and describe the tissue-specific expression of its mRNA, the enzyme characteristics of the 3 beta HSD IV protein, and the structural and functional relationships it has to other 3 beta HSD isoforms previously characterized in the mouse and rat. The predicted amino acid sequence of mouse 3 beta HSD IV is 77% and 73% identical to that of mouse 3 beta HSD I and III, respectively. Comparison of the nucleotide and amino acid sequences of the four isoforms characterized to date show that 3 beta HSD IV is more distantly related to I, II, and III than these three forms are to each other. 3 beta HSD IV mRNA is only expressed in mouse kidney. In situ hybridization of mouse kidney indicates that expression is found only in the cortex and appears to be associated with the proximal tubules. Transiently expressed 3 beta HSD IV protein could not convert the delta 5-3 beta-hydroxysteroids, pregnenolone or dehydroepiandrosterone, to their respective delta 4-3-<em>ketosteroids</em>, progesterone or androstenedione, nor did it have the capacity to convert 5 alpha-androstane-3 beta, <em>17</em> beta-diol to dihydrotestosterone, characteristic enzymatic activities of expressed mouse 3 beta HSD I and III. 3 beta HSD IV could only catalyze the conversion of dihydrotestosterone to 5 alpha-androstanediol in the presence of the cofactor NADPH. Thus, 3 beta HSD IV is a 3-<em>ketosteroid</em> reductase rather than a 3 beta-hydroxysteroid dehydrogenase/isomerase despite its homology to the other members of the 3 beta HSD family. Mouse 3 beta HSD IV is functionally and structurally most closely related to rat 3 beta HSD III, an isoform expressed predominantly in male rat liver.

1. The aim of this study was to investigate the mechanism by which amphetamine exerts its inhibitory effect on testicular interstitial cells of male rats. 2. Administration of amphetamine (10(-12)-10(-6) M) in vitro resulted in a dose-dependent inhibition of both basal and human chorionic gonadotropin (hCG, 0.05 iu ml(-1))-stimulated release of testosterone. 3. Amphetamine (10(-9) M) enhanced the basal and hCG-increased levels of adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation in vitro (P<0.05) in rat testicular interstitial cells. 4. Administration of SQ22536, an adenylyl cyclase inhibitor, decreased the basal release (P<0.05) of testosterone in vitro and abolished the inhibitory effect of amphetamine. 5. Nifedipine (10(-6) M) alone decreased the secretion of testosterone (P<0.01) but it failed to modify the inhibitory action of amphetamine (10(-10)-10(-6) M). 6. Amphetamine (10(-10)-10(-6) M) significantly (P<0.05 or P<0.01) decreased the activities of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450c<em>17</em>, and <em>17</em>-<em>ketosteroid</em> reductase (<em>17</em>-KSR) as indicated by thin-layer chromatography. (t.l.c.). 7. These results suggest that increased cyclic AMP production, decreased Ca2+ channel activity and decreased activities of 3beta-HSD, P450c<em>17</em>, and <em>17</em>-KSR are involved in the inhibition of testosterone production induced by the administration of amphetamine.

<em>17</em>β-Hydroxysteroid dehydrogenases (<em>17</em>β-HSDs) catalyze the reduction of <em>17</em>-<em>ketosteroids</em> and the oxidation of <em>17</em>β-hydroxysteroids to regulate the production of androgens and estrogens. Among them, <em>17</em>β-HSD type 3 (HSD<em>17</em>B3) is expressed almost exclusively in testicular Leydig cells and contributes to development of male sexual characteristics by converting androstenedione (A4) to testosterone (T). Mutations of HSD<em>17</em>B3 genes cause a 46,XY disorder of sexual development (46,XY DSD) as a result of low T production. Therefore, the evaluation of <em>17</em>β-HSD3 enzymatic activity is important for understanding and diagnosing this disorder. We adapted a method that easily evaluates enzymatic activity of <em>17</em>β-HSD3 by quantifying the conversion from A4 to T using androgen receptor (AR)-mediated transactivation. HEK293 cells were transduced to express human HSD<em>17</em>B3, and incubated medium containing A4. Depending on the incubation time with HSD<em>17</em>B3-expressing cells, the culture media progressively increased luciferase activities in CV-1 cells, transfected with the AR expression vector and androgen-responsive reporter. Culture medium from HSD<em>17</em>B1 and HSD<em>17</em>B5-expressing cells also increased the luciferase activities. This system is also applicable to detect the conversion of 11-ketoandrostenedione to 11-ketotestosterone by HSD<em>17</em>B3. Establishment of HEK293 cells expressing various missense mutations in the HSD<em>17</em>B3 gene associated with 46,XY DSD revealed that this system is effective to evaluate the enzymatic activities of mutant proteins.

The authors determined the amounts of free and total <em>17</em>-hydroxycorticosteroids (<em>17</em>-OHCS) and <em>17</em>-<em>ketosteroids</em> (<em>17</em>-KS) in the urine of 11 women in whom epileptic seizures occurred most frequently on the 2nd and 1st day before menstruation and in 7 women with seizures showing no such correlation with the menstrual cycle. A statistically significant decrease of <em>17</em>-KS was demonstrated in women with premenstrual seizures. In both groups of women the amounts of free <em>17</em>-OHCS were near the lower normal range, while the amounts of total <em>17</em>-OHCS were reduced and the obtained results compared with the accepted normal values showed statistically significant differences (p less than 0.01). The obtained results are, however, insufficient for assuming that these hormones play an important role in the mechanism of seizure release in premenstrual period. The authors think, on the other hand, that the statistically significant fall in <em>17</em>-KS in women with premenstrual seizures may play some role among other factors in the mechanism of seizure release.

Antibodies were raised against 3-fluoro-4-aza-estradiol-<em>17</em>-hemisuccinate to elicit abzymes capable of isomerizing delta 5-3-<em>ketosteroids</em>. The hapten was designed to present a planar A ring showing some analogy with the intermediate dienol and a polarity capable of inducing catalytic groups. Antibodies binding the hapten tightly were cloned and purified from either ascites or hybridoma supernatants. Catalytic activity was tested with androst-5-ene-3,<em>17</em>-dione. Among the five different monoclonal antibodies binding the hapten, one proved to enhance significantly the substrate isomerization rate (kcat/kuncat = 830). Initial rates followed Michaelis kinetics and the activity was competitively inhibited by the hapten.

Polycystic ovaries were found in a 16-year-old female with congenital absence of vagina, male-like external genitalia, and congenital adrenal hyperplasia. Masculinization was sufficiently severe to cause the patient to be reared as a male. Biochemical studies of ovarian tissue revealed hyperactivity and an imbalance of enzyme systems concerned with steroid-hormone biosynthesis, which led to production of large amounts of androgens. The pathway towards estrogens was preserved but less efficient than normal. Urinary steroid metabolites before and after hysterectomy and bilateral salpingo-oophorectomy revealed an absence of Porter-Silber chromogens and tetrahydrocortisone. Excretion of aldosterone was normal and that of corticosterone slightly higher than normal. The patterns of urinary <em>17</em>-<em>ketosteroids</em>, pregnanediol, pregnanetriol and pregnanetriolone were similar to those commonly seen in congenital adrenal hyperplasia with steroid 21-hydroxylase deficiency. Urinary estrogens after panhysterectomy were low, being in the post-menopausal range. The pathogenesis of polycystic ovaries and their possible contribution to masculinization are discussed.